Skeletal muscle biochemical adaptations to exercise training in miniature swine

McAllister, Richard M., Brian L. Reiter, John F. Amann, andM. Harold Laughlin. Skeletal muscle biochemical adaptations toexercise training in miniature swine. J. Appl.Physiol. 82(6): 1862-1868, 1997.The primarypurpose of this study was to test the hypothesis that enduranceexercise training induces increased oxidative capacity in porcineskeletal muscle. To test this hypothesis, female miniature swine wereeither trained by treadmill running 5 days/wk over 16-20 wk (Trn;n = 35) or pen confined (Sed;n = 33). Myocardialhypertrophy, lower heart rates during submaximal stages of a maximaltreadmill running test, and increased running time to exhaustion duringthat test were indicative of training efficacy. A variety of skeletalmuscles were sampled and subsequently assayed for the enzymes citratesynthase (CS), 3-hydroxyacyl-CoA dehydrogenase, and lactatedehydrogenase and for antioxidant enzymes. Fiber type composition of arepresentative muscle was also determined histochemically. The largestincrease in CS activity (62%) was found in the gluteus maximus muscle(Sed, 14.7 ± 1.1 µmol · min¹ · g¹;Trn, 23.9 ± 1.0; P < 0.0005).Muscles exhibiting increased CS activity, however, were locatedprimarily in the forelimb; ankle and knee extensor and respiratorymuscles were unchanged with training. Only two muscles exhibited higher3-hydroxyacyl-CoA dehydrogenase activity in Trn compared with Sed.Lactate dehydrogenase activity was unchanged with training, as wereactivities of antioxidant enzymes. Histochemical analysis of thetriceps brachii muscle (long head) revealed lower type IIB fibernumbers in Trn (Sed, 42 ± 6%; Trn, 10 ± 4;P < 0.01) and greater type IID/Xfiber numbers (Sed, 11 ± 2; Trn, 22 ± 3;P < 0.025). These findingsindicate that porcine skeletal muscle adapts to endurance exercisetraining in a manner similar to muscle of humans and other animalmodels, with increased oxidative capacity. Specificmuscles exhibiting these adaptations, however, differ between theminiature swine and other species.

     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

Skeletal muscle hypertrophy in response to isometric, lengthening, and shortening training bouts of equivalent duration.

Movements generated by muscle contraction generally include periods of muscle shortening and lengthening as well as force development in the absence of external length changes (isometric). However, in the specific case of resistance exercise training, exercises are often intentionally designed to emphasize one of these modes. The purpose of the present study was to objectively evaluate the relative effectiveness of each training mode for inducing compensatory hypertrophy. With the use of a rat model with electrically stimulated (sciatic nerve) contractions, groups of rats completed 10 training sessions in 20 days. Within each training session, the duration of the stimulation was equal across the three modes. Although this protocol provided equivalent durations of duty cycle, the torque integral for the individual contractions varied markedly with training mode such that lengthening > isometric > shortening. The results indicate that the hypertrophy response did not track the torque integral with mass increases of isometric by 14%, shortening by 12%, and lengthening by 11%. All three modes of training resulted in similar increases in total muscle DNA and RNA. Isometric and shortening but not lengthening mode training resulted in increased muscle insulin-like growth factor I mRNA levels. These results indicate that relatively pure movement mode exercises result in similar levels of compensatory hypertrophy that do not necessarily track with the total amount of force generated during each contraction.     

Autophagic response to exercise training in skeletal muscle with age

Autophagy, a highly conserved quality control mechanism, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. During aging, the efficiency of autophagic degradation declines, and intracellular waste products accumulate. Therefore, in this study, we tested the hypothesis that skeletal muscle from old mice would have decreased autophagosome formation when compared to the muscle from young mice. We also examined whether autophagic regulatory events differ between muscle fiber types and in response to exercise in aged male mice. The extensor digitorum longus (EDL) and gastrocnemius muscles were studied in young and old ICR mice. Exercise was performed by allowing the mice to run on a treadmill with a 5° incline at 16.4 m/min for 40 min/day, 5 days/week for 8 weeks after a 1-week adaptation period. Our results indicated that the levels of microtubule-associated protein 1b light chain 3, a marker of autophagosome formation, were lower in both the EDL and the gastrocnemius muscle of old mice compared to those young mice. To identify the factors related to the changes observed, the expression of autophagy regulatory proteins was examined in the EDL and gastrocnemius muscles. Beclin-1, autophagy-related gene 7 (ATG7), and lysosome-associated membrane protein were found to be lower in the EDL and gastrocnemius muscles of old mice compared to those in the young mice, then Beclin-1, ATG7, and muscle-specific RING finger protein-1 upregulated after regular exercise. Moreover, the muscle weight/body weight was significantly increased only in the gastrocnemius muscle of the old trained mice. These data suggest that autophagy regulatory events are attenuated in old skeletal muscle. However, this effect is upregulated when animals are subjected to exercise training.     

Skeletal muscle glucose transport in obese Zucker rats after exercise training

Exercise training has been found to reduce the muscle insulin resistance of the obese Zucker rat (fa/fa). The purpose of the present study was to determine whether this reduction in muscle insulin resistance was associated with an improvement in the glucose transport process and if it was fiber-type specific. Rats were randomly assigned to a sedentary or training group. Training consisted of treadmill running at 18 m/min up an 8% grade, 1.5 h/day, 5 days/wk, for 6-8 wk. The rate of muscle glucose transport was assessed in the absence of insulin and in the presence of a physiological (0.15 mU/ml), a submaximal (1.50 mU/ml), and a maximal (15.0 mU/ml) insulin concentration by determining the rate of 3-O-methyl-D-glucose (3-OMG) accumulation during hindlimb perfusion. The average 3-OMG transport rate of the red gastrocnemii (fast-twitch oxidative-glycolytic fibers) was significantly higher in the trained compared with the sedentary obese rats in the absence of insulin and in the presence of the three insulin concentrations. Significant improvements in 3-OMG transport were also observed in the plantarii (mixed fibers) of trained obese rats in the presence of 0, 0.15, and 15.0 mU/ml insulin. Training appeared to have little effect on the insulin-stimulated 3-OMG transport of the soleus (slow-twitch oxidative fibers) or white gastrocnemius (fast-twitch glycolytic fibers). The results suggest that the improvement in the muscle insulin resistance of the obese Zucker rat after moderate endurance training was associated with an improvement in the glucose transport process but that it was fiber-type specific.     

Skeletal muscle metabolic and ionic adaptations during intense exercise following sprint training in humans.

The effects of sprint training on muscle metabolism and ion regulation during intense exercise remain controversial. We employed a rigorous methodological approach, contrasting these responses during exercise to exhaustion and during identical work before and after training. Seven untrained men undertook 7 wk of sprint training. Subjects cycled to exhaustion at 130% pretraining peak oxygen uptake before (PreExh) and after training (PostExh), as well as performing another posttraining test identical to PreExh (PostMatch). Biopsies were taken at rest and immediately postexercise. After training in PostMatch, muscle and plasma lactate (Lac(-)) and H(+) concentrations, anaerobic ATP production rate, glycogen and ATP degradation, IMP accumulation, and peak plasma K(+) and norepinephrine concentrations were reduced (P<0.05). In PostExh, time to exhaustion was 21% greater than PreExh (P<0.001); however, muscle Lac(-) accumulation was unchanged; muscle H(+) concentration, ATP degradation, IMP accumulation, and anaerobic ATP production rate were reduced; and plasma Lac(-), norepinephrine, and H(+) concentrations were higher (P<0.05). Sprint training resulted in reduced anaerobic ATP generation during intense exercise, suggesting that aerobic metabolism was enhanced, which may allow increased time to fatigue.     

Effects of training, exercise and diet on muscle glycolysis and liver gluconeogenesis.

Trained and untrained rats were fed either a control, high-fat, or high-carbohydrate diet and then sacrificed in either a rested or exhausted state. The $\text{in vitro}$ activity of several muscle glycolytic and liver gluconeogenic enzymes was measured. Muscle hexokinase, phosphorylase, and phosphofructokinase activities were increased after training. Hexokinase was decreased in exhausted rats. Phosphorylase and phosphofructokinase were increased in untrained-exhausted rats but were unchanged in trained-exhausted rats. Liver pyruvate carboxylase and phosphoenolpyruvate carboxykinase activities were increased in trained-rested rats fed a high-fat diet. In trained-exhausted rats phosphoenolpyruvate carboxykinase activity was increased regardless of diet fed. Blood glucose was decreased in trained-exhausted rats, but it was increased in untrained-exhausted rats. Plasma glucocorticoid level was increased in exhausted rats. This study showed that training was associated with an increased muscle glycolytic capacity. Training was also related to the ability of liver to increase phosphoenolpyruvate carboxykinase activity during exercise, thereby increasing gluconeogenic capacity.     

Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia.

Gene expression of vascular endothelial growth factor (VEGF), and to a lesser extent of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF), has been found to increase in rat skeletal muscle after a single exercise bout. In addition, acute hypoxia augments the VEGF mRNA response to exercise, which suggests that, if VEGF is important in muscle angiogenesis, hypoxic training might produce greater capillary growth than normoxic training. Therefore, we examined the effects of exercise training (treadmill running at the same absolute intensity) in normoxia and hypoxia (inspired O(2) fraction = 0.12) on rat skeletal muscle capillarity and on resting and postexercise gene expression of VEGF, its major receptors (flt-1 and flk-1), TGF-beta(1), and bFGF. Normoxic training did not alter basal or exercise-induced VEGF mRNA levels but produced a modest twofold increase in bFGF mRNA (P < 0.05). Rats trained in hypoxia exhibited an attenuated VEGF mRNA response to exercise (1.8-fold compared 3.4-fold with normoxic training; P < 0.05), absent TGF-beta(1) and flt-1 mRNA responses to exercise, and an approximately threefold (P < 0.05) decrease in bFGF mRNA levels. flk-1 mRNA levels were not significantly altered by either normoxic or hypoxic training. An increase in skeletal muscle capillarity was observed only in hypoxically trained rats. These data show that, whereas training in hypoxia potentiates the adaptive angiogenic response of skeletal muscle to a given absolute intensity of exercise, this was not evident in the gene expression of VEGF or its receptors when assessed at the end of training.     

Skeletal muscle energy metabolism during prolonged, fatiguing exercise.

A depletion of phosphocreatine (PCr), fall in the total adenine nucleotide pool (TAN = ATP + ADP + AMP), and increase in TAN degradation products inosine 5'-monophosphate (IMP) and hypoxanthine are observed at fatigue during prolonged exercise at 70% maximal O(2) uptake in untrained subjects [J. Baldwin, R. J. Snow, M. F. Carey, and M. A. Febbraio. Am. J. Physiol. 277 (Regulatory Integrative Comp. Physiol. 46): R295-R300, 1999]. The present study aimed to examine whether these metabolic changes are also prevalent when exercise is performed below the blood lactate threshold (LT). Six healthy, untrained humans exercised on a cycle ergometer to voluntary exhaustion at an intensity equivalent to 93 +/- 3% of LT ( approximately 65% peak O(2) uptake). Muscle biopsy samples were obtained at rest, at 10 min of exercise, approximately 40 min before fatigue (F-40 =143 +/- 13 min), and at fatigue (F = 186 +/- 31 min). Glycogen concentration progressively declined (P < 0.01) to very low levels at fatigue (28 +/- 6 mmol glucosyl U/kg dry wt). Despite this, PCr content was not different when F-40 was compared with F and was only reduced by 40% when F was compared with rest (52. 8 +/- 3.7 vs. 87.8 +/- 2.0 mmol/kg dry wt; P < 0.01). In addition, TAN concentration was not reduced, IMP did not increase significantly throughout exercise, and hypoxanthine was not detected in any muscle samples. A significant correlation (r = 0.95; P < 0. 05) was observed between exercise time and glycogen use, indicating that glycogen availability is a limiting factor during prolonged exercise below LT. However, because TAN was not reduced, PCr was not depleted, and no correlation was observed between glycogen content and IMP when glycogen stores were compromised, fatigue may be related to processes other than those involved in muscle high-energy phosphagen metabolism.     

Skeletal muscle protein anabolic response to resistance exercise and essential amino acids is delayed with aging.

Skeletal muscle loss during aging leads to an increased risk of falls, fractures, and eventually loss of independence. Resistance exercise is a useful intervention to prevent sarcopenia; however, the muscle protein synthesis (MPS) response to resistance exercise is less in elderly compared with young subjects. On the other hand, essential amino acids (EAA) increase MPS equally in both young and old subjects when sufficient EAA is ingested. We hypothesized that EAA ingestion following a bout of resistance exercise would stimulate anabolic signaling and MPS similarly between young and old men. Each subject ingested 20 g of EAA 1 h following leg resistance exercise. Muscle biopsies were obtained before and 1, 3, and 6 h after exercise to measure the rate of MPS and signaling pathways that regulate translation initiation. MPS increased early in young (1-3 h postexercise) and later in old (3-6 h postexercise). At 1 h postexercise, ERK1/2 MNK1 phosphorylation increased and eIF2alpha phosphorylation decreased only in the young. mTOR signaling (mTOR, S6K1, 4E-BP1, eEF2) was similar between groups at all time points, but MNK1 phosphorylation was lower at 3 h and AMP-activated protein kinase-alpha (AMPKalpha) phosphorylation was higher in old 1-3 h postexercise. We conclude that the acute MPS response after resistance exercise and EAA ingestion is similar between young and old men; however, the response is delayed with aging. Unresponsive ERK1/2 signaling and AMPK activation in old muscle may be playing a role in the delayed activation of MPS. Notwithstanding, the combination of resistance exercise and EAA ingestion should be a useful strategy to combat sarcopenia.     

Skeletal muscle RAS and exercise performance

A local renin-angiotensin system (RAS) may be suggested by evidence of gene expression of RAS components within the tissue as well as physiological responsiveness of this gene expression. This review will focus on the evidence supporting the existence of the constituent elements of a physiologically functional paracrine muscle RAS. The effect of local skeletal muscle RAS on human exercise performance will be explored via its relation with pharmacological intervention and genetic studies.The most likely configuration of the muscle RAS is a combination of in situ synthesis and uptake from the circulation of RAS components. A reduction in angiotensin-converting enzyme (ACE) activity reverses the decline in physical performance due to peripheral muscle factors in those with congestive heart failure and may halt or slow decline in muscle strength in elderly women. Genetic studies suggest that increased ACE and angiotensin II (Ang II) mediate greater strength gains perhaps via muscle hypertrophy whereas lower ACE levels and reduced bradykinin (BK) degradation mediate enhanced endurance performance perhaps via changes in substrate availability, muscle fibre type and efficiency.     

Physical training in man. Skeletal muscle metabolism in relation to muscle morphology and running ability.

The metabolic and morphologic adaptation to physical training in skeletal muscle tissue of eleven middle-aged, physically untrained men was studied. Muscle biopsies were taken from the vastus lateralis before, after 8 weeks and after 6 months of physical training for analysis of metabolic and morphologic variables. Glucose tolerance test indicated increased insulin sensitivity after 6 months of physical training. The activities of glycogen phosphorylase, hexokinase and glucose-6-P-dehydrogenase were increased but other enzymes involved in glycogen turnover and glycolysis were unchanged after 6 months of physical traning. The activities of citrate synthase and cytochrome-c-oxidase, representing the oxidative capacity were significantly increased already after 8 weeks of physical training. The incorporation rate of palmitate-carbon into CO2 and triglycerides increased, and the incorporation rate of leucine-carbon into CO2 decreased with 6 months of physical training. The fiber diameter of both Type 1- and Type 2-fibers increased, while the mitochondrial volume increased predominantly in Type 2-fibers. Significant correlations were found between metabolic, physiologic and morphologic variables before and after physical training. The results indicate an increased oxidative capacity, mainly located to Type 2-fibers, and an increased utilization of fatty acids in response to this type of physical training.     

Skeletal muscle nNOS mu protein content is increased by exercise training in humans

The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOS mu. Exercise training increases nNOS mu protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOS mu protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOS mu protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOS mu protein was approximately 60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOS mu protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOS mu protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOS mu protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOS mu expression in untrained individuals, due to uniform increases of nNOS mu within types I, IIa, and IIx fibers.     

Skeletal muscle adaptation to exercise: a century of progress.

Skeletal muscle physiology and biochemistry is an established field with Nobel Prize-winning scientists, dating back to the 1920s. Not until the mid to late 1960s did there appear a major focus on physiological and biochemical training adaptations in skeletal muscle. The study of adaptations to exercise training reveals a wide range of integrative approaches, from the systemic to the molecular level. Advances in our understanding of training adaptations have come in waves caused by the introduction of new experimental approaches. Research has revealed that exercise can be effective at preventing and/or treating some of the most common chronic diseases of the latter half of the 20th century. Endurance-trained muscle is more effective at clearing plasma triglyceride, glucose, and free fatty acids. However, at the present time, most of the mechanisms underlying the adaptation of human skeletal muscle to exercise still remain to be discovered. Little is known about the regulatory factors (e.g., trans-acting proteins or signaling pathways) directly modulating the expression of exercise-responsive genes. Because so many potential physiological and biochemical signals change during exercise, it will be an important challenge in the next century to move beyond "correlational studies" and to identify responsible mechanisms. Skeletal muscle metabolic adaptations may prove to be a critical component to preventing diseases such as coronary heart disease, type 2 diabetes, and obesity. Therefore, training studies have had an impact on setting the stage for a potential "preventive medicine reformation" in a society needing a return to a naturally active lifestyle of our ancestors.     

Skeletal muscle proteolysis in response to short-term unloading in humans.

Skeletal muscle atrophy is evident after muscle disuse, unloading, or spaceflight and results from decreased protein content as a consequence of decreased protein synthesis, increased protein breakdown or both. At this time, there are essentially no human data describing proteolysis in skeletal muscle undergoing atrophy on Earth or in space, primarily due to lack of valid and accurate methodology. This particular study aimed at assessing the effects of short-term unloading on the muscle contractile proteolysis rate. Eight men were subjected to 72-h unilateral lower limb suspension (ULLS) and intramuscular interstitial levels of the naturally occurring proteolytic tracer 3-methylhistidine (3MH) were measured by means of microdialysis before and on completion of this intervention. The 3MH concentration following 72-h ULLS (2.01 +/- 0.22 nmol/ml) was 44% higher (P < 0.05) than before ULLS (1.56 +/- 0.20 nmol/ml). The present experimental model and the employed method determining 3MH in microdialysates present a promising tool for monitoring skeletal muscle proteolysis or metabolism of specific muscles during conditions resulting in atrophy caused by, e.g., disuse and real or simulated microgravity. This study provides evidence that the atrophic processes are evoked rapidly and within 72 h of unloading and suggests that countermeasures should be employed in the early stages of space missions to offset or prevent muscle loss during the period when the rate of muscle atrophy is the highest.     

Angiogenic growth factor expression in rat skeletal muscle in response to exercise training

Angiogenesis occurs in skeletal muscle in response to exercise training. To gain insight into the regulation of this process, we evaluated the mRNA expression of factors implicated in angiogenesis over the course of a training program. We studied sedentary control (n = 17) rats and both sedentary (n = 18) and exercise-trained (n = 48) rats with bilateral femoral artery ligation. Training consisted of treadmill exercise (4 times/day, 1-24 days). Basal mRNA expression in sedentary control muscle was inversely related to muscle vascularity. Angiogenesis was histologically evident in trained white gastrocnemius muscle by day 12. Training produced initial three- to sixfold increases in VEGF, VEGF receptors (KDR and Flt), the angiopoietin receptor (Tie-2), and endothelial nitric oxide synthase mRNA, which dissipated before the increase in capillarity, and a substantial (30- to 50-fold) but transient upregulation of monocyte chemoattractant protein 1 mRNA. These results emphasize the importance of early events in regulating angiogenesis. However, we observed a sustained elevation of the angiopoietin 2-to-angiopoietin 1 ratio, suggesting continued vascular destabilization. The response to exercise was (in general) tempered in high-oxidative muscles. These findings place importance on cellular events coupled to the onset of angiogenesis.     

Skeletal muscle intracellular PO(2) assessed by myoglobin desaturation: response to graded exercise.

The relationship between skeletal muscle intracellular PO(2) (iPO(2)) and progressive muscular work has important implications for the understanding of O(2) transport and utilization. Presently there is debate as to whether iPO(2) falls progressively with increasing O(2) demand or reaches a plateau from moderate to maximal metabolic demand. Thus, using (1)H magnetic resonance spectroscopy of myoglobin (Mb), we studied cellular oxygenation during progressive single-leg knee extensor exercise from unweighted to 100% of maximal work rate in six active human subjects. In all subjects, the Mb peak at 73 ppm was not visible at rest, whereas the peak was small or indistinguishable from the noise in the majority of subjects during progressive exercise from unweighted to 50-60% of maximum work rate. In contrast, beyond this exercise intensity, a Mb peak of consistent magnitude was discernible in all subjects. When a Mb half saturation of 3.2 Torr was used, the calculated skeletal muscle PO(2) was variable before 60% of maximum work rate but in general was relatively high (>18 Torr, the measurable PO(2) with the poorest signal-to-noise ratio, in the majority of cases), whereas beyond this exercise intensity iPO(2) fell to a relatively uniform and invariant level of 3.8 +/- 0.5 Torr across all subjects. These results do not support the concept of a progressive linear fall in iPO(2) across increasing work rates. Instead, this study documents variable but relatively high iPO(2) from rest to moderate exercise and again confirms that from 50-60% of maximum work rate iPO(2) reaches a plateau that is then invariant with increasing work rate.