The effect of exercise intensity on skeletal muscle AMP-activated protein kinase (AMPK) signaling and substrate metabolism was examined in eight men cycling for 20 min at each of three sequential intensities: low (40 ؎ 2% VO 2 peak), medium (59 ؎ 1% VO 2 peak), and high (79 ؎ 1% VO 2 peak). Muscle free AMP/ATP ratio only increased at the two higher exercise intensities (P < 0.05). AMPK ␣1 (1.5-fold) and AMPK ␣2 (5-fold) activities increased from low to medium intensity, with AMPK ␣2 activity increasing further from medium to high intensity. The upstream AMPK kinase activity was substantial at rest and only increased 50% with exercise, indicating that, initially, signaling through AMPK did not require AMPK kinase posttranslational modification. Acetyl-CoA carboxylase (ACC)-␤ phosphorylation was sensitive to exercise, increasing threefold from rest to low intensity, whereas neuronal NO synthase (nNOS) phosphorylation was only observed at the higher exercise intensities. Glucose disappearance (tracer) did not increase from rest to low intensity, but increased sequentially from low to medium to high intensity. Calculated fat oxidation increased from rest to low intensity in parallel with ACC␤ phosphorylation, then declined during high intensity. These results indicate that ACC␤ phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.

We examined the effect of short-term exercise training on skeletal muscle AMP-activated protein kinase (AMPK) signalling and muscle metabolism during prolonged exercise in humans. Eight sedentary males completed 120 min of cycling at 66 ± 1%V O 2 peak , then exercise trained for 10 days, before repeating the exercise bout at the same absolute workload. Participants rested for 72 h before each trial while ingesting a high carbohydrate diet (HCHO). Exercise training significantly (P < 0.05) attenuated exercise-induced increases in skeletal muscle free AMP : ATP ratio and glucose disposal and increased fat oxidation. Exercise training abolished the 9-fold increase in AMPK α2 activity observed during pretraining exercise. Since training increased muscle glycogen content by 93 ± 12% (P < 0.01), we conducted a second experiment in seven sedentary male participants where muscle glycogen content was essentially matched pre-and post-training by exercise and a low CHO diet (LCHO; post-training muscle glycogen 52 ± 7% less than in HCHO, P < 0.001). Despite the difference in muscle glycogen levels in the two studies we obtained very similar results. In both studies the increase in ACCβ Ser 221 phosphorylation was reduced during exercise after training. In conclusion, there is little activation of AMPK signalling during prolonged exercise following short-term exercise training suggesting that other factors are important in the regulation of glucose disposal and fat oxidation under these circumstances. It appears that muscle glycogen is not an important regulator of AMPK activation during exercise in humans when exercise is begun with normal or high muscle glycogen levels.

. Progressive increase in human skeletal muscle AMPK␣2 activity and ACC phosphorylation during exercise. Am J Physiol Endocrinol Metab 282: E688-E694, 2002; 10.1152/ajpendo.00101.2001.-The effect of prolonged moderate-intensity exercise on human skeletal muscle AMP-activated protein kinase (AMPK)␣1 and -␣2 activity and acetyl-CoA carboxylase (ACC␤) and neuronal nitric oxide synthase (nNOS) phosphorylation was investigated. Seven active healthy individuals cycled for 30 min at a workload requiring 62.8 Ϯ 1.3% of peak O2 consumption (V O2 peak) with muscle biopsies obtained from the vastus lateralis at rest and at 5 and 30 min of exercise. AMPK␣1 activity was not altered by exercise; however, AMPK␣2 activity was significantly (P Ͻ 0.05) elevated after 5 min (ϳ2-fold), and further elevated (P Ͻ 0.05) after 30 min (ϳ3-fold) of exercise. ACC␤ phosphorylation was increased (P Ͻ 0.05) after 5 min (ϳ18-fold compared with rest) and increased (P Ͻ 0.05) further after 30 min of exercise (ϳ36-fold compared with rest). Increases in AMPK␣2 activity were significantly correlated with both increases in ACC␤ phosphorylation and reductions in muscle glycogen content. Fat oxidation tended (P ϭ 0.058) to increase progressively during exercise. Muscle creatine phosphate was lower (P Ͻ 0.05), and muscle creatine, calculated free AMP, and free AMP-to-ATP ratio were higher (P Ͻ 0.05) at both 5 and 30 min of exercise compared with those at rest. At 30 min of exercise, the values of these metabolites were not significantly different from those at 5 min of exercise. Phosphorylation of nNOS was variable, and despite the mean doubling with exercise, statistically significance was not achieved (P ϭ 0.304). Western blots indicated that AMPK␣2 was associated with both nNOS and ACC␤ consistent with them both being substrates of AMPK␣2 in vivo. In conclusion, AMPK␣2 activity and ACC␤ phosphorylation increase progressively during moderate exercise at ϳ60% of V O2 peak in humans, with these responses more closely coupled to muscle glycogen content than muscle AMP/ ATP ratio. adenosine monophosphate-activated protein kinase; acetylcoenzyme A carboxylase-␤; neuronal nitric oxide synthase; prolonged exercise; humans AMP-ACTIVATED PROTEIN KINASE (AMPK) plays an important role in coordinating metabolism with a number of physiological processes (16,22). In rat skeletal muscle, activation of AMPK appears to lead to enhanced fat oxidation and glucose uptake (3,19,25). In transgenic mice expressing a dominant negative mutant of AMPK, there is impaired glucose uptake in response to both contraction and hypoxia (26). There are two isoforms of AMPK, AMPK␣1 and -␣2, expressed in skeletal muscle. Maximal sprint exercise [ϳ200% peak O 2 consumption (V O 2 peak )] over 30 s in humans causes a large increase in the calculated free AMP and free AMP-to-ATP ratio (AMP/ATP) (28) and activates both AMPK␣1 and -␣2 (6). AMPK␣1 is not activated during lower intensity exercise such as at 50 or 70% V O 2 peak , or after 5 min of exercise at 90% V O 2 peak after 55 min of cy...

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.

There appears to be little benefit from ingesting water during intense 1-h cycling exercise in mild environmental conditions since such ingestion has no significant effect on HR, body temperature, plasma volume, plasma electrolytes, or performance.