Exercise-induced regulation of muscular Na+-K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type

It is investigated if exercise-induced mRNA changes cause similar protein expression changes of Na(+)-K(+) pump isoforms (α(1), α(2), β(1), β(2)), FXYD1, and Na(+)/K(+) exchanger (NHE1) in rat skeletal muscle. Expression was evaluated (n = 8 per group) in soleus and extensor digutorum longus after 1 day, 3 days, and 3 wk (5 sessions/wk) of either sprint (4 × 3-min sprint + 1-min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of soleus α(2) (4.9 ± 0.8-fold; P < 0.05), β(2) (13.2 ± 4.4-fold; P < 0.001), and NHE1 (12.0 ± 3.1-fold; P < 0.01). Two hours after sprint exercise, protein expression normalized to control samples was higher on day 3 than day 1 for soleus α(1) (41 ± 18% increase vs. 15 ± 8% reduction; P < 0.05), α(2) (64 ± 35% increase vs. 37 ± 12% reduction; P < 0.05), β(1) (17 ± 21% increase vs. 14 ± 29% reduction; P < 0.05), and FXYD1 (35 ± 16% increase vs. 13 ± 10% reduction; P < 0.05). In contrast, on day 3, soleus α(1) (0.1 ± 0.1-fold; P < 0.001), α(2) (0.2 ± 0.1-fold; P < 0.001), β(1) (0.4 ± 0.1-fold; P < 0.05), and β(2)-mRNA (2.9 ± 1.7-fold; P < 0.001) expression was lower than after exercise on day 1. After 3 wk of training, no change in protein expression relative to control existed. In conclusion, increased expression of Na(+)-K(+) pump subunits, FXYD1 and NHE1 after 3 days exercise training does not appear to be an effect of increased constitutive mRNA levels. Importantly, sprint exercise can reduce mRNA expression concomitant with increased protein expression.     

Effect of exhaustive ultra-endurance exercise in muscular glycogen and both Alpha1 and Alpha2 Ampk protein expression in trained rats

Glycogen is the main store of readily energy in skeletal muscle and plays a key role in muscle function, demonstrated by the inability to sustain prolonged high-intensity exercise upon depletion of these glycogen stores. With prolonged exercise, glycogen depletion occurs and 5′-AMP-activated protein kinase (AMPK), a potent regulator of muscle metabolism and gene expression, is activated promoting molecular signalling that increases glucose uptake by muscular skeletal cells. The aim of this study was primarily to determine the effect of ultra-endurance exercise on muscle glycogen reserves and secondly to verify the influence of this type of exercise on AMPK protein expression. Twenty-four male Wistar rats, 60 days old, were divided into four experimental groups: sedentary, sedentary exhausted (SE), endurance trained (T) and endurance trained exhausted (TE). The animals ran for 10 to 90 min/day, 5 days/week, for 12 weeks to attain trained status. Rats were killed immediately after the exhaustion protocol, which consisted of running on a treadmill (at approximately 60 % V _max until exhaustion). Optical density of periodic acid-Schiff was detected and glycogen depletion observed predominantly in type I muscle fibres of the TE group and in both type I and II muscle fibres in the SE group. Plasma glucose decreased only in the TE group. Hepatic glycogen was increased in T group and significantly depleted in TE group. AMPK protein expression was significantly elevated in TE and T groups. In conclusion, acute exhaustive ultra-endurance exercise promoted muscle glycogen depletion. It seems that total AMPK protein and gene expression is more influenced by status training.     

补糖和/或刺五加对大鼠运动后骨骼肌细胞AMPK蛋白表达的影响

目的:研究补糖和刺五加对大鼠运动后骨骼肌细胞的AMP激活蛋白激酶(AMPK)蛋白表达的影响及其恢复期的时相性变化。方法:128只SD大鼠大鼠随机分为训练对照组(C组)、训练补糖组(G组)、训练补刺五加皂甙组(A组)和训练补糖补刺五加皂甙组(GA组)四大组,补糖和刺五加均在运动后0.5 h内灌胃给予。根据运动前和运动后不同时间(0 h,4 h,12 h)采样,共分为16小组(n=8)。采用Western blot方法分析骨骼肌的AMPK蛋白含量。结果:①运动后骨骼肌的AMPK蛋白表达量上调,运动后即刻最高(209.23±21.32),随后逐渐恢复;②补药显著地提高了机体在消耗糖原运动后即刻和4 h后的股四头肌AMPK蛋白含量(225.11±20.58和186.31±15.26vs195.19±13.31和157.11±16.43),运动后12 h两组间没有差异;③补糖对骨骼肌AMPK的蛋白表达量的影响没有统计学意义;④补糖同时补药可提高运动后即刻和4 h后的股四头肌AMPK蛋白含量(217.96±19.25和191.86±14.69),但是运动后12h反而低于对照组(121.89±15.23vs137.92±16.01)。结论:运动可激活骨骼肌细胞AMPK,补充刺五加皂甙可上调运动后的AMPK蛋白表达,补糖则没有影响。     

5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle

The 5'-AMP-activated protein kinase (AMPK) is proposed to be involved in signaling pathways leading to adaptations in skeletal muscle in response to both a single exercise bout and exercise training. This study investigated the effect of endurance training on protein content of catalytic (alpha1, alpha2) and regulatory (beta1, beta2 and gamma1, gamma2, gamma3) subunit isoforms of AMPK as well as on basal AMPK activity in human skeletal muscle. Eight healthy young men performed supervised one-legged knee extensor endurance training for 3 wk. Muscle biopsies were obtained before and 15 h after training in both legs. In response to training the protein content of alpha1, beta2 and gamma1 increased in the trained leg by 41, 34, and 26%, respectively (alpha1 and beta2 P < 0.005, gamma1 P < 0.05). In contrast, the protein content of the regulatory gamma3-isoform decreased by 62% in the trained leg (P = 0.01), whereas no effect of training was seen for alpha2, beta1, and gamma2. AMPK activity associated with the alpha1- and the alpha2-isoforms increased in the trained leg by 94 and 49%, respectively (both P < 0.005). In agreement with these observations, phosphorylation of alpha-AMPK-(Thr172) and of the AMPK target acetyl-CoA carboxylase-beta(Ser221) increased by 74 and 180%, respectively (both P < 0.001). Essentially similar results were obtained in four additional subjects studied 55 h after training. This study demonstrates that protein content and basal AMPK activity in human skeletal muscle are highly susceptible to endurance exercise training. Except for the increase in gamma1 protein, all observed adaptations to training could be ascribed to local contraction-induced mechanisms, since they did not occur in the contralateral untrained muscle.     

β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg(-1)·min(-1) peak O(2) uptake, 26.7 ± 2.0 kg/m(2) body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O(2) uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [(2)H(5)]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ～2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content.     

Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested or ran for 30 or 60 min on a treadmill (22 m/min, 10% slope) were sacrificed immediately after exercise or after 60 min recovery either in the fasted state or after oral gavage with glucose (3g/kg body weight). Exercise decreased muscle and liver glycogen substantially. Hypothalamic total or alpha2-associated AMPK activity and phosphorylation state of the AMPK substrate acetyl-CoA carboxylase were not changed significantly immediately following treadmill running or during fed or fasted recovery. Plasma ghrelin increased (P<0.05) by 40% during exercise whereas the concentration of PYY was unchanged. In recovery, glucose feeding increased plasma glucose and insulin concentrations whereas ghrelin and PYY decreased to (ghrelin) or below (PPY) resting levels. It is concluded that 1h of strenuous exercise in rats does not elicit significant changes in hypothalamic AMPK activity despite an increase in plasma ghrelin. Thus, changes in energy metabolism during or after exercise are likely not coordinated by changes in hypothalamic AMPK activity.     

Exercise training blunts oxidative stress in sickle cell trait carriers

The aim of this study was to analyze the effects of exercise training on oxidative stress in sickle cell trait carriers. Plasma levels of oxidative stress [advanced oxidation protein products (AOPP), protein carbonyl, malondialdehyde (MDA), and nitrotyrosine], antioxidant markers [catalase, glutathione peroxidase (GPX), and superoxide dismutase (SOD)], and nitrite and nitrate (NOx) were assessed at baseline, immediately following a maximal exercise test (T(ex)), and during recovery (T(1h), T(2h), T(24h)) in trained (T: 8 h/wk minimum) and untrained (U: no regular physical activity) sickle cell trait (SCT) carriers or control (CON) subjects (T-SCT, n = 10; U-SCT, n = 8; T-CON, n = 11; and U-CON, n = 11; age: 23.5 ± 2.2 yr). The trained subjects had higher SOD activities (7.6 ± 5.4 vs. 5.2 ± 2.1 U/ml, P = 0.016) and lower levels of AOPP (142 ± 102 vs. 177 ± 102 μM, P = 0.028) and protein carbonyl (82.1 ± 26.0 vs. 107.3 ± 30.6 nm/ml, P = 0.010) than the untrained subjects in response to exercise. In response to exercise, U-SCT had a higher level of AOPP (224 ± 130 vs. 174 ± 121 μM, P = 0.012), nitrotyrosine (127 ± 29.1 vs.70.6 ± 46.6 nM, P = 0.003), and protein carbonyl (114 ± 34.0 vs. 86.9 ± 26.8 nm/ml, P = 0.006) compared with T-SCT. T-SCT had a higher SOD activity (8.50 ± 7.2 vs. 4.30 ± 2.5 U/ml, P = 0.002) and NOx (28.8 ± 11.4 vs. 14.6 ± 7.0 μmol·l(-1)·min(-1), P = 0.003) in response to exercise than U-SCT. Our data indicate that the overall oxidative stress and nitric oxide response is improved in exercise-trained SCT carriers compared with their untrained counterparts. These results suggest that physical activity could be a viable method of controlling the oxidative stress. This could have a beneficial impact because of its involvement in endothelial dysfunction and subsequent vascular impairment in hemoglobin S carriers.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

Effects of exercise training on airway closure in asthmatics

We previously reported that responsiveness to methacholine (Mch) in the absence of deep inspiration (DI) decreased in healthy subjects after a short course of exercise training. We assessed whether a similar beneficial effect of exercise on airway responsiveness could occur in asthmatics. Nine patients (male/female: 3/6; mean age ± SD: 24 ± 2 yr) with mild untreated asthma [forced expiratory volume in 1 s (FEV(1)): 100 ± 7.4% pred; FEV(1)/vital capacity (VC): 90 ± 6.5%] underwent a series of single-dose Mch bronchoprovocations in the absence of DI in the course of a 10-wk training rowing program (6 h/wk of submaximal and maximal exercise), at baseline (week 0), and at week 5 and 10. The single-dose Mch was established as the dose able to induce ≥15% reduction in inspiratory vital capacity (IVC) and was administered to each subject at every challenge occasion. Five asthmatics (male/female: 1/4; mean age ± SD: 26 ± 3 yr) with similar baseline lung function (FEV(1): 102 ± 7.0% predicted; FEV(1)/VC: 83 ± 6.0%; P = 0.57 and P = 0.06, respectively) not participating in the exercise training program served as controls. In the trained group, the Mch-induced reduction in IVC from baseline was 22 ± 10% at week 0, 13 ± 11% at week 5 (P = 0.03), and 11 ± 8% at week 10 (P = 0.028). The Mch-induced reduction in FEV(1) did not change with exercise (P = 0.69). The reduction in responsiveness induced by exercise was of the same magnitude of that previously obtained in healthy subjects (50% with respect to pretraining). Conversely, Mch-induced reduction in IVC in controls remained unchanged after 10 wk (%reduction IVC at baseline: 21 ± 20%; after 10 wk: 29 ± 14%; P = 0.28). This study indicates that a short course of physical training is capable of reducing airway responsiveness in mild asthmatics.     

Interleukin-6 release from human skeletal muscle during exercise: relation to AMPK activity.

We tested the hypothesis that IL-6 release from muscle during exercise may be related to muscle activity of 5'-AMP-activated protein kinase (AMPK). Eight healthy, well-trained young men completed two 60-min trials on a bicycle ergometer at 70% of their peak oxygen uptake in either a glycogen-depleted or a glycogen-loaded state. IL-6 was released from the leg already after 10 min of exercise in the glycogen-depleted state, whereas no significant release was observed at any time in the loaded state. Nevertheless, plasma IL-6 increased similarly in the two trials from approximately 0.8 pg/ml at rest to approximately 4.5 pg/ml after 60 min of exercise. Activity of alpha1-AMPK (160%) and alpha2-AMPK (145%) was increased at rest in the glycogen-depleted compared with the loaded situation. During exercise, alpha1-AMPK activity did not change from resting levels in both trials, whereas alpha2-AMPK activity increased only in the glycogen-depleted state. After 60 min of exercise in the glycogen-depleted state, individual values of alpha2-AMPK activity correlated significantly (r = 0.87, P < 0.006) with individual values of IL-6 release as well as with average IL-6 release over the entire 60 min (r = 0.86, P < 0.006). The present data are compatible with a role for AMPK in IL-6 release during exercise or a role for IL-6 in activating AMPK. Alternatively, both AMPK and IL-6 are independent sensors of a low muscle glycogen concentration during exercise. In addition, leg release of IL-6 cannot alone explain the increase in plasma IL-6 during exercise.     

Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.     

AS160 phosphorylation is associated with activation of alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK trimeric complex in skeletal muscle during exercise in humans

We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% VO2 peak, n=8), 20 min (80% VO2 peak, n=11), 2 min (110% of peak work rate, n=9), or 30 s (maximal sprint, n=10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P=0.06) and further at 90 min of exercise (120%, P<0.05). alpha2beta2gamma3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas alpha2beta2gamma1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. alpha2beta2gamma1-AMPK activity and AS160 phosphorylation correlated positively (r2=0.55). In exercise trials 2, 3, and 4, alpha2beta2gamma3-AMPK activity but neither AS160 phosphorylation nor alpha2beta2gamma1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.     

The role of exercise intensity in the bone metabolic response to an acute bout of weight-bearing exercise

We compared the effects of exercise intensity (EI) on bone metabolism during and for 4 days after acute, weight-bearing endurance exercise. Ten males [mean ± SD maximum oxygen uptake (Vo(2max)): 56.2 ± 8.1 ml·min(-1)·kg(-1)] completed three counterbalanced 8-day trials. Following three control days, on day 4, subjects completed 60 min of running at 55%, 65%, and 75% Vo(2max). Markers of bone resorption [COOH-terminal telopeptide region of collagen type 1 (β-CTX)] and formation [NH(2)-terminal propeptides of procollagen type 1 (P1NP), osteocalcin (OC), bone-alkaline phosphatase (ALP)], osteoprotegerin (OPG), parathyroid hormone (PTH), albumin-adjusted calcium (ACa), phosphate (PO(4)), and cortisol were measured during and for 3 h after exercise and on four follow-up days (FU1-FU4). At 75% Vo(2max), β-CTX was not significantly increased from baseline by exercise but was higher compared with 55% (17-19%, P < 0.01) and 65% (11-13%, P < 0.05) Vo(2max) in the first hour postexercise. Concentrations were decreased from baseline in all three groups by 39-42% (P < 0.001) at 3 h postexercise but not thereafter. P1NP increased (P < 0.001) during exercise only, while bone-ALP was increased (P < 0.01) at FU3 and FU4, but neither were affected by EI. PTH and cortisol increased (P < 0.001) with exercise at 75% Vo(2max) only and were higher (P < 0.05) than at 55% and 65% Vo(2max) during and immediately after exercise. The increases (P < 0.001) in OPG, ACa, and PO(4) with exercise were not affected by EI. Increasing EI from 55% to 75% Vo(2max) during 60 min of running resulted in higher β-CTX concentrations in the first hour postexercise but had no effect on bone formation markers. Increased bone-ALP concentrations at 3 and 4 days postexercise suggest a beneficial effect of this type of exercise on bone mineralization. The increase in OPG was not influenced by exercise intensity, whereas PTH was increased at 75% Vo(2max) only, which cannot be fully explained by changes in serum calcium or PO(4) concentrations.     

Resistance exercise load does not determine training-mediated hypertrophic gains in young men

We have reported that the acute postexercise increases in muscle protein synthesis rates, with differing nutritional support, are predictive of longer-term training-induced muscle hypertrophy. Here, we aimed to test whether the same was true with acute exercise-mediated changes in muscle protein synthesis. Eighteen men (21 ± 1 yr, 22.6 ± 2.1 kg/m(2); means ± SE) had their legs randomly assigned to two of three training conditions that differed in contraction intensity [% of maximal strength (1 repetition maximum)] or contraction volume (1 or 3 sets of repetitions): 30%-3, 80%-1, and 80%-3. Subjects trained each leg with their assigned regime for a period of 10 wk, 3 times/wk. We made pre- and posttraining measures of strength, muscle volume by magnetic resonance (MR) scans, as well as pre- and posttraining biopsies of the vastus lateralis, and a single postexercise (1 h) biopsy following the first bout of exercise, to measure signaling proteins. Training-induced increases in MR-measured muscle volume were significant (P < 0.01), with no difference between groups: 30%-3 = 6.8 ± 1.8%, 80%-1 = 3.2 ± 0.8%, and 80%-3= 7.2 ± 1.9%, P = 0.18. Isotonic maximal strength gains were not different between 80%-1 and 80%-3, but were greater than 30%-3 (P = 0.04), whereas training-induced isometric strength gains were significant but not different between conditions (P = 0.92). Biopsies taken 1 h following the initial resistance exercise bout showed increased phosphorylation (P < 0.05) of p70S6K only in the 80%-1 and 80%-3 conditions. There was no correlation between phosphorylation of any signaling protein and hypertrophy. In accordance with our previous acute measurements of muscle protein synthetic rates a lower load lifted to failure resulted in similar hypertrophy as a heavy load lifted to failure.     

Aging and acute exercise enhance free radical generation in rat skeletal muscle.

Reactive oxygen species (ROS) are implicated in the mechanism of biological aging and exercise-induced oxidative damage. The present study examined the effect of an acute bout of exercise on intracellular ROS production, lipid and protein peroxidation, and GSH status in the skeletal muscle of young adult (8 mo, n = 24) and old (24 mo, n = 24) female Fischer 344 rats. Young rats ran on a treadmill at 25 m/min and 5% grade until exhaustion (55.4 +/- 2.7 min), whereas old rats ran at 15 m/min and 5% grade until exhaustion (58.0 +/- 2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of ROS and other intracellular oxidants production in the homogenate of deep vastus lateralis, was 77% (P < 0.01) higher in rested old vs. young rats. Exercise increased DCFH oxidation by 38% (P < 0.09) and 50% (P < 0.01) in the young and old rats, respectively. DCFH oxidation in isolated deep vastus lateralis mitochondria with site 1 substrates was elevated by 57% (P < 0.01) in old vs. young rats but was unaltered with exercise. Significantly higher DCFH oxidation rate was also found in aged-muscle mitochondria (P < 0.01), but not in homogenates, when ADP, NADPH, and Fe(3+) were included in the assay medium without substrates. Lipid peroxidation in muscle measured by malondialdehyde content showed no age effect, but was increased by 20% (P < 0.05) with exercise in both young and old rats. Muscle protein carbonyl formation was unaffected by either age or exercise. Mitochondrial GSH/ GSSG ratio was significantly higher in aged vs. young rats (P < 0.05), whereas exercise increased GSSG content and decreased GSH/GSSG in both age groups (P < 0.05). These data provided direct evidence that oxidant production in skeletal muscle is increased in old age and during prolonged exercise, with both mitochondrial respiratory chain and NADPH oxidase as potential sources. The alterations of muscle lipid peroxidation and mitochondrial GSH status were consistent with these conclusions.     

AMPK-dependent and independent actions of P2X7 in regulation of mitochondrial and lysosomal functions in microglia

Background

P2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases. Since mitochondrial function in microglia is highly associated with microglial functions in controlling neuronal plasticity and brain homeostasis, we interested to explore the roles of P2X7 in mitochondrial and lysosomal functions as well as mitophagy in microglia.

Methods

P2X7^?/? bone marrow-derived macrophages (BMDM), primary microglia and BV-2 immortalized microglial cells were used to detect the particular protein expression by immunoblotting. Mitochondrial reactive oxygen species (mitoROS), intracellular calcium, mitochondrial mass and lysosomal integrity were examined by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics and mitophagy after P2X7 activation.

Results

In primary microglia, BV-2 microglial cells and BMDM, P2X7 agonist BzATP triggered AMPK activation and LC3II accumulation through reactive oxygen species (ROS) and CaMKKII pathways, and these effects were abolished by P2X7 antagonist A438079 and P2X7 deficiency. Moreover, we detected the dramatic decreases of mitochondrial OCR and mass following P2X7 activation. AMPK inhibition by compound C or AMPK silencing reversed the P2X7 actions in reduction of mitochondrial mass, induction of mitochondrial fission and mitophagy, but not in uncoupling of mitochondrial respiration. Interestingly, we found that P2X7 activation induced nuclear translocation of TFEB via an AMPK-dependent pathway and led to lysosomal biogenesis. Mimicking the actions of BzATP, nigericin also induced ROS-dependent AMPK activation, mitophagy, mitochondrial fission and respiratory inhibition. Longer exposure of BzATP induced cell death, and this effect was accompanied by the lysosomal instability and was inhibited by autophagy and cathepsin B inhibitors.

Conclusion

Altogether ROS- and CaMKK-dependent AMPK activation is involved in P2X7-mediated mitophagy, mitochondrial dynamics and lysosomal biogenesis in microglial cells, which is followed by cytotoxicity partially resulting from mitophagy and cathepsin B activation.

     

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of l-[ring-(13)C(6)]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTOR(Ser2448) was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1(Thr389) was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exercise-induced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.