5'-AMP-activated protein kinase (AMPK) has been suggested to be a 'metabolic master switch' regulating various aspects of muscle glucose and fat metabolism. In isolated rat skeletal muscle, glucose suppresses the activity of AMPK and in human muscle glycogen loading decreases exercise-induced AM PK activation. We hypothesized that oral glucose ingestion during exercise would attenuate muscle AMPK activation. Nine male subjects performed two bouts of one-legged knee-extensor exercise at 60% of maximal workload. The subjects were randomly assigned to either Consume a glucose containing drink or a placebo drink during the two trials. Muscle biopsies were taken from the vastus lateralis before and after 2 h of exercise. Plasma glucose was higher (6.0 +/- 0.2 vs. 4.9 +/- 0.1 mmol L-1, P < 0.001), whereas glycerol (44.8 +/- 7.8 vs. 165.7 +/- 22.3 mu mol L-1), and free fatty acid (169.3 +/- 9.5 vs. 1161 +/- 144.9 mu mol L-1) concentrations were lower during the glucose compared to the placebo trial (both P < 0.001). Calculated fat oxidation was lower during the glucose trial (0.17 +/- 0.02 vs. 0.25 +/- 0.03 g min(-1), P < 0.001). Activation of alpha 2-AMPK was attenuated in the glucose trial compared to the placebo trial (0.24 +/-0.07 vs. 0.46 +/- 0.14 mu mol mg(-1) min(-1), P = 0.03), whereas the alpha 1-AMPK activity was not different between trials or affected by exercise. AMPK and the downstream target of AMPK, acetyl-CoA carboxylase-beta, were phosphorylated as a response to exercise, but neither was significantly different between the two trials. We conclude that oral glucose ingestion attenuates the exercise-induced activation of alpha 2-AMPK, bringing further support for a fuel-sensing role of AMPK in skeletal muscle. (c) 2006 Elsevier Inc. All rights reserved.