Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)     

nNOS regulation of skeletal muscle fatigue and exercise performance

Neuronal nitric oxide synthases (nNOS) are Ca²⁺/calmodulin-activated enzymes that synthesize the gaseous messenger nitric oxide (NO). nNOSμ and the recently described nNOSβ, both spliced nNOS isoforms, are important enzymatic sources of NO in skeletal muscle, a tissue long considered to be a paradigmatic system for studying NO-dependent redox signaling. nNOS is indispensable for skeletal muscle integrity and contractile performance, and deregulation of nNOSμ signaling is a common pathogenic feature of many neuromuscular diseases. Recent evidence suggests that both nNOSμ and nNOSβ regulate skeletal muscle size, strength, and fatigue resistance, making them important players in exercise performance. nNOSμ acts as an activity sensor and appears to assist skeletal muscle adaptation to new functional demands, particularly those of endurance exercise. Prolonged inactivity leads to nNOS-mediated muscle atrophy through a FoxO-dependent pathway. nNOS also plays a role in modulating exercise performance in neuromuscular disease. In the mdx mouse model of Duchenne muscular dystrophy, defective nNOS signaling is thought to restrict contractile capacity of working muscle in two ways: loss of sarcolemmal nNOSμ causes excessive ischemic damage while residual cytosolic nNOSμ contributes to hypernitrosylation of the ryanodine receptor, causing pathogenic Ca²⁺ leak. This defect in Ca²⁺ handling promotes muscle damage, weakness, and fatigue. This review addresses these recent advances in the understanding of nNOS-dependent redox regulation of skeletal muscle function and exercise performance under physiological and neuromuscular disease conditions.     

Contraction regulation of Akt in rat skeletal muscle.

The protein serine/threonine kinase Akt/protein kinase B has been recognized as a critical signaling mediator for multiple cell systems. The function of Akt in skeletal muscle is not well understood, and whether contractile activity stimulates Akt activity has been controversial. In the current study, contraction in situ, induced via sciatic nerve stimulation, significantly increased Akt Ser(473) phosphorylation in multiple muscle types including the extensor digitorum longus (13-fold over basal), plantaris (5.8-fold), red gastrocnemius (4.7-fold), white gastrocnemius (3.3-fold), and soleus (1.6-fold). In addition to increasing phosphorylation, contraction in situ significantly increased the activity of all three Akt isoforms (Akt1 > Akt2 > Akt3) with maximal activation occurring at 2.5 min and returning to base line with 15 min of contraction. Akt phosphorylation and activity were also increased when isolated muscles were contracted in vitro in the absence of systemic factors, although to a much lesser extent. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 fully inhibited contraction-stimulated Akt phosphorylation and activity but did not diminish contraction-stimulated glycogen synthase kinase-3 phosphorylation and glycogen synthase activity. These results demonstrate that contraction increases Akt phosphorylation and activity in skeletal muscle and that this stimulation is rapid, transient, muscle fiber type-specific, and wortmannin- and LY294002-inhibitable. Akt signaling is not necessary for the regulation of glycogen synthase activity in contracting skeletal muscle.     

Adaptations in rat skeletal muscle following long-term resistance exercise training

The purpose of this investigation was to determine whether long-term, heavy resistance training would cause adaptations in rat skeletal muscle structure and function. Ten male Wistar rats (3 weeks old) were trained to climb a 40-cm vertical ladder (4 days/week) while carrying progressively heavier loads secured to their tails. After 26 weeks of training the rats were capable of lifting up to 800 g or 140% of their individual body mass for four sets of 12–15 repetitions per session. No difference in body mass was observed between the trained rats and age-matched sedentary control rats. Absolute and relative heart mass were greater in trained rats than control rats. When expressed relative to body mass, the mass of the extensor digitorum longus (EDL) and soleus muscles was greater in trained rats than control rats. No difference in absolute muscle mass or maximum force-producing capacity was evident in either the EDL or soleus muscles after training, although both muscles exhibited an increased resistance to fatigue. Individual fibre hypertrophy was evident in all four skeletal muscles investigated, i.e. EDL, soleus, plantaris and rectus femoris muscles of trained rats, but muscle fibre type proportions within each of the muscles tested remained unchanged. Despite an increased ability of the rats to lift progressively heavier loads, this heavy resistance training model did not induce gross muscle hypertrophy nor did it increase the force-producing capacity of the EDL or soleus muscles. Accepted: 17 September 1997     

Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro‐ and anti‐inflammatory cytokines, and recently, it has been recognized as an important source of interleukin‐6 (IL‐6). Acute physical exercise is known to induce a pro‐inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro‐ and anti‐inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL‐6, TNF‐α, IL‐1β and IL‐10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week^?1 for 8 weeks (60% VO_2max). Detection of IL‐6, TNF‐α, IL‐1β and IL‐10 protein expression was carried out by ELISA. We found decreased expression of IL‐1β, IL‐6, TNF‐α and IL‐10 (28%, 27%, 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL‐1β, TNF‐α and IL‐10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL‐6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8‐week moderate‐intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright © 2009 John Wiley & Sons, Ltd.     

Akt signaling in skeletal muscle: regulation by exercise and passive stretch

Akt/protein kinase B is a serine/threonine kinase that has emerged as a critical signaling component for mediating numerous cellular responses. Contractile activity has recently been demonstrated to stimulate Akt signaling in skeletal muscle. Whether physiological exercise in vivo activates Akt is controversial, and the initiating factors that result in the stimulation of Akt during contractile activity are unknown. In the current study, we demonstrate that treadmill running exercise of rats using two different protocols (intermediate high or high-intensity exhaustive exercise) significantly increases Akt activity and phosphorylation in skeletal muscle composed of various fiber types. To determine if Akt activation during contractile activity is triggered by mechanical forces applied to the skeletal muscle, isolated skeletal muscles were incubated and passively stretched. Passive stretch for 10 min significantly increased Akt activity (2-fold) in the fast-twitch extensor digitorum longus (EDL) muscle. However, stretch had no effect on Akt in the slow-twitch soleus muscle, although there was a robust phosphorylation of the stress-activated protein kinase p38. Similar to contraction, stretch-induced Akt activation in the EDL was fully inhibited in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas glycogen synthase kinase-3 (GSK3) phosphorylation was only partially inhibited. Stretch did not cause dephosphorylation of glycogen synthase on GSK3-targeted sites in the absence or presence of wortmannin. We conclude that physiological exercise in vivo activates Akt in multiple skeletal muscle fiber types and that mechanical tension may be a part of the mechanism by which contraction activates Akt in fast-twitch muscles.     

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.     

Bdnf expression in rat skeletal muscle after acute or repeated exercise

Brain derived growth factor (BDNF) gene of rat has a complex structure: at least four 5' untranslated exons regulated by different promoters and one 3' exon containing the encoding region. BDNF is expressed by skeletal muscles in an activity-dependent manner. In this study, BDNF mRNA was analysed by RT-PCR in the soleus muscle following a single (acute) session of running or a training of five days of running (repetitive exercise). Moreover, the expression of the exons was quantitatively analysed by real time RT-PCR. Finally, muscle BDNF protein level was evaluated by western blotting. BDNF mRNA was found to increase over the second day after acute exercise; on the other hand, two peaks (2 and 24 hours after the last session, respectively) in BDNF mRNA level were found after repetitive exercise, but it was similar to that of controls 6 hours after the last session. BDNF protein level progressively increased also after the mRNA went back to the basal level, so suggesting that it cumulates within the cell after acute exercise, whereas it followed the mRNA level time course after repetitive exercise. These results point to the following conclusions: BDNF mRNA is up-regulated by activity, but this response is delayed to the second day after acute exercise; repetitive exercise transiently depresses the expression of BDNF mRNA, so that the over-expression due to the previous day's exercise completely disappears 6 hours after the last exercise session.     

Populations of rat skeletal muscle mitochondria after exercise and immobilization

We slightly modified an existing procedure (Palmer et al., J. Biol. Chem. 252: 8731-8739, 1977) to isolate two distinct populations of mitochondria from rat skeletal muscle; initial brief Polytron homogenization released the subsarcolemmal mitochondria, and brief exposure of the resultant intact myofibrils to the proteolytic enzyme, Nagarse, extracted the intermyofibrillar mitochondria. The intermyofibrillar mitochondria differed from the subsarcolemmal mitochondr. ia by higher state III respiration measurements and enzymatic activities. These two populations of mitochondria were then isolated from the gastrocnemius muscle that had been induced to perform different amounts of contractile activity. The endurance training program of daily running significantly increased state III respiration and respiratory control index in the subsarcolemmal mitochondria, but the program did not increase these measurements in the intermyofibrillar mitochondria. In addition, 2 days of hindlimb immobilization resulted in a significant decrease in state II respiration and the respiratory control index of the subsarcolemmal mitochondria; however, immobilization did not affect the intermyofibrillar mitochondria. These measurements suggest that the subsarcolemmal mitochondria adapt in response to chronic changes in the level of contractile activity.     

Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tail-suspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tail-suspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.     

Changes in the rat skeletal muscle proteome induced by moderate-intensity endurance exercise

The adaptation of skeletal muscle to endurance exercise has not previously been investigated using proteomic techniques. Such work could improve our understanding and generate novel information regarding the effects of exercise. Plantaris muscles were investigated from rats exercised on treadmills at 70-75% peak oxygen uptake (V O(2)peak) for 30 min, 4 days per week for 5 weeks or sedentary controls. Analysis of 2-D gels matched 187 spots across control and exercised muscles and 80 proteins corresponding to 40 gene products were identified by MALDI-ToF MS. Exercise increased the animals' V O(2)peak by 14% and altered the expression of 15 spots consistent with a shift from glycolysis toward greater fatty-acid oxidation. The majority of differentially expressed gene products were present as multi-spot series of similar M(r) but different pI. Mitochondrial aconitase focused to 5 spots, 2 spots (pI 7.6 and 7.7) decreased (57%) whereas the pI 8.0 spot increased (51%) and was found to contain protein carbonyls. This adaptation may be related to exercise-induced oxidative stress and translocation of aconitase to mitochondrial DNA. In conclusion, proteomic techniques simultaneously demonstrated well-established effects, and identified novel changes not previously associated with the adaptation of muscle to exercise.     

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.     

Neural regulation of differentiation of rat skeletal muscle cell types

Summary Three monoclonal antibodies (LM5, F2 and F39) to the fast class of myosin heavy chain (MHC) were used to study the effect of denervation on the differentiation of muscle cell types in some rat skeletal muscles. Antibody LM5 in immunocytochemical investigations did not stain any myotubes during early fetal development but presumptive fast muscle cells started to stain during later fetal development. Unlike antibody LM5, antibodies F2 and F39 stained all myotubes during fetal development. The suppression of fast myosin heavy chains recognised in presumptive slow muscle cells was observed within 1–2 days after birth with antibody F39 but not until 10–14 days after birth with antibody F2. The emergence of subsets of fast muscle fibre types in rat extensor digitorum longus (EDL) and tibialis anteri (TA) detectable by F39 and F2 antibodies was not observed until 2–3 weeks after birth. Denervation of developing muscles led to marked changes in the expression of myosins identified by these antibodies.     

Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise

Intracellular mechanisms regulating fat oxidation were investigated in human skeletal muscle during exercise. Eight young, healthy, moderately trained men performed bicycle exercise (60 min, 65% peak O2 consumption) on two occasions, where they ingested either 1) a high-carbohydrate diet (H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to alter muscle glycogen content as well as to induce, respectively, low and high rates of fat oxidation. Leg fat oxidation was 122% higher during exercise in L-CHO than in H-CHO (P < 0.001). In keeping with this, the activity of alpha2-AMP-activated protein kinase (alpha2-AMPK) was increased twice as much in L-CHO as in H-CHO (P < 0.01) at 60 min of exercise. However, acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both conditions (P < 0.05). Pyruvate dehydrogenase activity was higher during exercise in H-CHO than in L-CHO (P < 0.01). In H-CHO only, the concentrations of acetyl-CoA and acetylcarnitine were increased (P < 0.001), and the concentration of free carnitine was decreased (P < 0.01), by exercise. The data suggest that a decrease in the concentration of malonyl-CoA, secondary to alpha2-AMPK activation and ACC inhibition (by phosphorylation), contributes to the increase in fat oxidation observed at the onset of exercise regardless of muscle glycogen levels. They also suggest that, with high muscle glycogen, the availability of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation.