Endurance training reduces the contraction-induced interleukin-6 mRNA expression in human skeletal muscle

Contracting skeletal muscle expresses large amounts of IL-6. Because 1) IL-6 mRNA expression in contracting skeletal muscle is enhanced by low muscle glycogen content, and 2) IL-6 increases lipolysis and oxidation of fatty acids, we hypothesized that regular exercise training, associated with increased levels of resting muscle glycogen and enhanced capacity to oxidize fatty acids, would lead to a less-pronounced increase of skeletal muscle IL-6 mRNA in response to acute exercise. Thus, before and after 10 wk of knee extensor endurance training, skeletal muscle IL-6 mRNA expression was determined in young healthy men (n = 7) in response to 3 h of dynamic knee extensor exercise, using the same relative workload. Maximal power output, time to exhaustion during submaximal exercise, resting muscle glycogen content, and citrate synthase and 3-hydroxyacyl-CoA dehydrogenase enzyme activity were all significantly enhanced by training. IL-6 mRNA expression in resting skeletal muscle did not change in response to training. However, although absolute workload during acute exercise was 44% higher (P < 0.05) after the training period, skeletal muscle IL-6 mRNA content increased 76-fold (P < 0.05) in response to exercise before the training period, but only 8-fold (P < 0.05, relative to rest and pretraining) in response to exercise after training. Furthermore, the exercise-induced increase of plasma IL-6 (P < 0.05, pre- and posttraining) was not higher after training despite higher absolute work intensity. In conclusion, the magnitude of the exercise-induced IL-6 mRNA expression in contracting human skeletal muscle was markedly reduced by 10 wk of training.     

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.     

Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle.

Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 muM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased alpha(2)-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and alpha(2)-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced alpha(2)-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced alpha(2)-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca(2+)-independent activation of ERK1/2 as well as Ca(2+)-dependent activation of CaMKII and AMPK.     

Resistance exercise enhances the molecular signaling of mitochondrial biogenesis induced by endurance exercise in human skeletal muscle

Combining endurance and strength training (concurrent training) may change the adaptation compared with single mode training. However, the site of interaction and the mechanisms are unclear. We have investigated the hypothesis that molecular signaling of mitochondrial biogenesis after endurance exercise is impaired by resistance exercise. Ten healthy subjects performed either only endurance exercise (E; 1-h cycling at ～65% of maximal oxygen uptake), or endurance exercise followed by resistance exercise (ER; 1-h cycling + 6 sets of leg press at 70-80% of 1 repetition maximum) in a randomized cross-over design. Muscle biopsies were obtained before and after exercise (1 and 3 h postcycling). The mRNA of genes related to mitochondrial biogenesis [(peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1)α, PGC-1-related coactivator (PRC)] related coactivator) and substrate regulation (pyruvate dehydrogenase kinase-4) increased after both E and ER, but the mRNA levels were about twofold higher after ER (P < 0.01). Phosphorylation of proteins involved in the signaling cascade of protein synthesis [mammalian target of rapamycin (mTOR), ribosomal S6 kinase 1, and eukaryotic elongation factor 2] was altered after ER but not after E. Moreover, ER induced a larger increase in mRNA of genes associated with positive mTOR signaling (cMyc and Rheb). Phosphorylation of AMP-activated protein kinase, acetyl-CoA carboxylase, and Akt increased similarly at 1 h postcycling (P < 0.01) after both types of exercise. Contrary to our hypothesis, the results demonstrate that ER, performed after E, amplifies the adaptive signaling response of mitochondrial biogenesis compared with single-mode endurance exercise. The mechanism may relate to a cross talk between signaling pathways mediated by mTOR. The results suggest that concurrent training may be beneficial for the adaptation of muscle oxidative capacity.     

Effect of endurance training on muscle TCA cycle metabolism during exercise in humans.

We tested the theory that links the capacity to perform prolonged exercise with the size of the muscle tricarboxylic acid (TCA) cycle intermediate (TCAI) pool. We hypothesized that endurance training would attenuate the exercise-induced increase in TCAI concentration ([TCAI]); however, the lower [TCAI] would not compromise cycle endurance capacity. Eight men (22 +/- 1 yr) cycled at approximately 80% of initial peak oxygen uptake before and after 7 wk of training (1 h/day, 5 days/wk). Biopsies (vastus lateralis) were obtained during both trials at rest, after 5 min, and at the point of exhaustion during the pretraining trial (42 +/- 6 min). A biopsy was also obtained at the end of exercise during the posttraining trial (91 +/- 6 min). In addition to improved performance, training increased (P < 0.05) peak oxygen uptake and citrate synthase maximal activity. The sum of four measured TCAI was similar between trials at rest but lower after 5 min of exercise posttraining [2.7 +/- 0.2 vs. 4.3 +/- 0.2 mmol/kg dry wt (P < 0.05)]. There was a clear dissociation between [TCAI] and endurance capacity because the [TCAI] at the point of exhaustion during the pretraining trial was not different between trials (posttraining: 2.9 +/- 0.2 vs. pretraining: 3.5 +/- 0.2 mmol/kg dry wt), and yet cycle endurance time more than doubled in the posttraining trial. Training also attenuated the exercise-induced decrease in glutamate concentration (posttraining: 4.5 +/- 0.7 vs. pretraining: 7.7 +/- 0.6 mmol/kg dry wt) and increase in alanine concentration (posttraining: 3.3 +/- 0.2 vs. pretraining: 5.6 +/- 0.3 mmol/kg dry wt; P < 0.05), which is consistent with reduced carbon flux through alanine aminotransferase. We conclude that, after aerobic training, cycle endurance capacity is not limited by a decrease in muscle [TCAI].     

Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer(473) and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 +/- 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased ( approximately 30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer(473) was approximately 50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer(473) and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer(473) on the improvement in insulin sensitivity requires further elucidation.     

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.     

Differential activation of mTOR signaling by contractile activity in skeletal muscle

The cellular mechanisms by which contractile activity stimulates skeletal muscle hypertrophy are beginning to be elucidated and appear to include activation of the phosphatidylinositol 3-kinase signaling substrate mammalian target of rapamycin (mTOR). We examined the time course and location of mTOR phosphorylation in response to an acute bout of contractile activity. Rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla), tibialis anterior (TA), and soleus (Sol) muscles from stimulated and control limbs were collected immediately or 6 h after stimulation. HFES resulted in mTOR phosphorylation immediately after (3.4 +/- 0.9-fold, P < 0.01) contractile activity in Pla, whereas TA was unchanged compared with controls. mTOR phosphorylation remained elevated in Pla (3.6 +/- 0.6-fold) and increased in TA (4.6 +/- 0.9-fold, P < 0.05) 6 h after HFES. Interestingly, mTOR activation occurred predominantly in fibers expressing type IIa but not type I myosin heavy chain isoform. Furthermore, HFES induced modest ribosomal protein S6 kinase phosphorylation immediately after exercise in Pla (0.4 +/- 0.1-fold, P < 0.05) but not TA and more markedly 6 h after in both Pla and TA (1.4 +/- 0.4-fold vs. 2.4 +/- 0.3-fold, respectively, P < 0.01). Akt/PKB phosphorylation was similar to controls at both time points. These results suggest that mTOR signaling is increased after a single bout of muscle contractile activity. Despite reports that mTOR is activated downstream of Akt/PKB, in this study, HFES induced mTOR signaling independent of Akt/PKB phosphorylation. Fiber type-dependent mTOR phosphorylation may be a molecular basis by which some fiber types are more susceptible to contraction-induced hypertrophy.     

Reactive hyperemia is not responsible for stimulating muscle protein synthesis following blood flow restriction exercise

Blood flow restriction (BFR) to contracting skeletal muscle during low-intensity resistance exercise training increases muscle strength and size in humans. However, the mechanism(s) underlying these effects are largely unknown. We have previously shown that mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis (MPS) are stimulated following an acute bout of BFR exercise. The purpose of this study was to test the hypothesis that reactive hyperemia is the mechanism responsible for stimulating mTORC1 signaling and MPS following BFR exercise. Six young men (24 ± 2 yr) were used in a randomized crossover study consisting of two exercise trials: low-intensity resistance exercise with BFR (BFR trial) and low-intensity resistance exercise with sodium nitroprusside (SNP), a pharmacological vasodilator infusion into the femoral artery immediately after exercise to simulate the reactive hyperemia response after BFR exercise (SNP trial). Postexercise mixed-muscle fractional synthetic rate from the vastus lateralis increased by 49% in the BFR trial (P < 0.05) with no change in the SNP trial (P > 0.05). BFR exercise increased the phosphorylation of mTOR, S6 kinase 1, ribosomal protein S6, ERK1/2, and Mnk1-interacting kinase 1 (P < 0.05) with no changes in mTORC1 signaling in the SNP trial (P > 0.05). We conclude that reactive hyperemia is not a primary mechanism for BFR exercise-induced mTORC1 signaling and MPS. Further research is necessary to elucidate the cellular mechanism(s) responsible for the increase in mTOR signaling, MPS, and hypertrophy following acute and chronic BFR exercise.     

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.     

Role of vitamin C and E supplementation on IL-6 in response to training

Vitamin C and E supplementation has been shown to attenuate the acute exercise-induced increase in plasma interleukin-6 (IL-6) concentration. Here, we studied the effect of antioxidant vitamins on the regulation of IL-6 expression in muscle and the circulation in response to acute exercise before and after high-intensity endurance exercise training. Twenty-one young healthy men were allocated into either a vitamin (VT; vitamin C and E, n = 11) or a placebo (PL, n = 10) group. A 1-h acute bicycling exercise trial at 65% of maximal power output was performed before and after 12 wk of progressive endurance exercise training. In response to training, the acute exercise-induced IL-6 response was attenuated in PL (P < 0.02), but not in VT (P = 0.82). However, no clear difference between groups was observed (group × training: P = 0.13). Endurance exercise training also attenuated the acute exercise-induced increase in muscle-IL-6 mRNA in both groups. Oxidative stress, assessed by plasma protein carbonyls concentration, was overall higher in the VT compared with the PL group (group effect: P < 0.005). This was accompanied by a general increase in skeletal muscle mRNA expression of antioxidative enzymes, including catalase, copper-zinc superoxide dismutase, and glutathione peroxidase 1 mRNA expression in the VT group. However, skeletal muscle protein content of catalase, copper-zinc superoxide dismutase, or glutathione peroxidase 1 was not affected by training or supplementation. In conclusion, our results indicate that, although vitamin C and E supplementation may attenuate exercise-induced increases in plasma IL-6 there is no clear additive effect when combined with endurance training.     

Arctigenin efficiently enhanced sedentary mice treadmill endurance

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.     

mTOR integrates amino acid- and energy-sensing pathways

The AMP-activated protein kinase (AMPK) exists as a heterotrimetric complex comprising a catalytic alpha subunit and non-catalytic beta and gamma subunits. Under conditions of hypoxia, exercise, ischemia, heat shock, and low glucose, AMPK is activated allosterically by rising cellular AMP and by phosphorylation of the catalytic alpha subunit. The mammalian target of rapamycin (mTOR) controls cellular functions in response to amino acids and growth factors. Recent reports including our study have demonstrated the possible interplay between mTOR and AMPK signaling pathways, supporting a model in which mitochondrial dysfunction caused by the mitochondrial inhibitors or ATP depletion inhibits activation of p70 S6 kinase alpha (p70alpha), a downstream effector of mTOR, by activating AMPK. Leucine may stimulate p70alpha phosphorylation via mTOR pathway, in part, by serving both as a mitochondrial fuel through oxidative carboxylation and an allosteric activation of glutamate dehydrogenase. This hypothesis may support an idea in which leucine modulates mTOR function, in part by regulating mitochondrial function and AMPK. Further understanding of the role of mTOR in coordinating amino acid- and energy-sensing pathways would provide new insights into relationship between nutrients and cellular functions.     

Central Exercise Action Increases the AMPK and mTOR Response to Leptin

AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exercise is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exercise ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest period. Intracerebroventricular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitor prevented IL-6-induced anorexia. Activators of AMPK and fasting increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitor of AMPK had the opposite effect. Furthermore, the reduction of AMPK and ACC phosphorylation and increase in phosphorylation of proteins involved in mTOR signal transduction, observed in the hypothalamus after leptin infusion, were more pronounced in both lean and diet-induced obesity rats after acute exercise. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in responsiveness to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Thus, the effects of leptin on the AMPK/mTOR pathway, potentiated by acute exercise, may contribute to appetite suppressive actions in the hypothalamus.     

Effect of aerobic training on forced expiratory airflow in exercising asthmatic humans

Pulmonary function after exercise was evaluated in 22 asthmatic subjects before and after a 36-session training sequence of aerobic exercise. Training did not change pulmonary function values, except for a small increase in maximal voluntary ventilation (P less than 0.02), which was attributed to respiratory muscle training. After aerobic training, both external work at a given heart rate and peak O2 consumption increased by 30 and 15%, respectively. At the same minute ventilation (VE), immediate postexercise forced expiratory airflow was higher after training (P less than 0.02), and reduction in forced expiratory airflow during the first 9 min postexercise was less after training (P less than 0.01). The posttraining airflow response to the pretraining work load was, as expected, less than the pretraining response (P less than 0.02). Although the difference in maximal-to-minimal airflow at the same VE was similar before and after training, the airflow increase accounted for 50% of the response after training compared with 16% of the pretraining response. Furthermore the strong negative correlation (P less than 0.01) between maximal and minimal airflow both pre- and posttraining indicates that exercise-induced bronchospasm (EIB) severity is, in part, determined by the degree of exercise-induced bronchodilation. We conclude that aerobic training significantly increases exercise-induced bronchodilation and diminishes EIB.     

Intensified exercise training does not alter AMPK signaling in human skeletal muscle

The AMP-activated protein kinase (AMPK) cascade has been linked to many of the acute effects of exercise on skeletal muscle substrate metabolism, as well as to some of the chronic training-induced adaptations. We determined the effect of 3 wk of intensified training (HIT; 7 sessions of 8 x 5 min at 85% Vo2 peak) in skeletal muscle from well-trained athletes on AMPK responsiveness to exercise. Rates of whole body substrate oxidation were determined during a 90-min steady-state ride (SS) pre- and post-HIT. Muscle metabolites and AMPK signaling were determined from biopsies taken at rest and immediately after exercise during the first and seventh HIT sessions, performed at the same (absolute) pre-HIT work rate. HIT decreased rates of whole body carbohydrate oxidation (P < 0.05) and increased rates of fat oxidation (P < 0.05) during SS. Resting muscle glycogen and its utilization during intense exercise were unaffected by HIT. However, HIT induced a twofold decrease in muscle [lactate] (P < 0.05) and resulted in tighter metabolic regulation, i.e., attenuation of the decrease in the PCr/(PCr + Cr) ratio and of the increase in [AMPfree]/ATP. Resting activities of AMPKalpha1 and -alpha2 were similar post-HIT, with the magnitude of the rise in response to exercise similar pre- and post-HIT. AMPK phosphorylation at Thr172 on both the alpha1 and alpha2 subunits increased in response to exercise, with the magnitude of this rise being similar post-HIT. Acetyl-coenzyme A carboxylase-beta phosphorylation was similar at rest and, despite HIT-induced increases in whole body rates of fat oxidation, did not increase post-HIT. Our results indicate that, in well-trained individuals, short-term HIT improves metabolic control but does not blunt AMPK signaling in response to intense exercise.     

Effects of endurance training on activity and expression of AMP-activated protein kinase isoforms in rat muscles

The effects of endurance training on the response of muscle AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) to moderate treadmill exercise were examined. In red quadriceps, there was a large activation of alpha 2-AMPK and inactivation of ACC in response to exercise. This response was greatly reduced after training, probably because of reduced metabolic stress. In white quadriceps, there were no effects of exercise on AMPK or ACC, but alpha 2-activity was higher after training because of increased phosphorylation of Thr(172). In soleus, there were small increases in alpha 2-activity during exercise that were not affected by training. The expression of all seven AMPK subunit isoforms was also examined. The beta 2- and gamma 2-isoforms were most highly expressed in white quadriceps, and gamma 3 was expressed in red quadriceps and soleus. There was a threefold increase in expression of gamma 3 after training in red quadriceps only. Our results suggest that gamma 3 might have a special role in the adaptation to endurance exercise in muscles utilizing oxidative metabolism.     

Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells

In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (approximately 3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (approximately 3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (approximately 4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.     

Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle.

With age, skeletal muscle experiences substantial atrophy and weakness. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans and animals is limited. In the present study, we assessed the ability of muscle contractile activity to activate cellular pathways involved in muscle cell growth and myogenesis in adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats. A single bout of rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla) and tibialis anterior (TA) muscles were assayed for mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70(S6K)), and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total protein either at baseline, immediately after, or 6 h after HFES. mTOR phosphorylation was elevated in Pla (1.3 +/- 0.3-fold, P < 0.05) immediately after HFES and to a lesser extent 6 h after HFES (0.6 +/- 0.1-fold, P < 0.05) in O rats. Post-HFES, p70(S6K) phosphorylation increased 1.2 +/- 0.3-fold in TA (P < 0.05) and remained elevated 6 h later (0.6 +/- 0.2-fold, P < 0.05) in O rats. ERK phosphorylation was lower in O rats immediately after exercise in both TA (11.1 +/- 2.9 vs. 2.1 +/- 0.5-fold, P < 0.05) and Pla (6.5 +/- 1.5 vs. 1.8 +/- 0.5-fold, P < 0.05) and returned to baseline by 6 h in both Y and O rats. Phosphorylation of mTOR, p70(S6K), and ERK1/2 are increased in skeletal muscle after a single bout of in situ muscle contractile activity in aged animals, and the response is less than that observed in adult animals. These observations suggest that the anabolic response to a single bout of contraction is attenuated with aging and may help explain the reduced capacity for hypertrophy in aged animals.