Abstract
AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKalpha2 subunit (alpha2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in alpha2-KD mice; expression of neuronal NOS (NOSmicro) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 +/- 8 and 50 +/- 7%, respectively, in skeletal muscle of alpha2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSmicro expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.
Highlights
Play a key role in the response to energetic stress [1, 2], because of its sensitivity to increased cellular AMP levels [3]
In mouse models in which AMPK␣2 protein expression and/or activity has been impaired, contractions performed in isolated skeletal muscle in vitro, ex vivo, or in situ have demonstrated that skeletal muscle glucose uptake (MGU) is normal [9, 10], partially impaired [11, 18], or ablated [19]
Activities of Specific Electron Transport Chain (ETC) Complexes Are Reduced in Skeletal Muscle of ␣2-KD Mice—The finding that exercise capacity, V O2peak, and ATP generation are impaired, changes in arterial lactate levels are accelerated in ␣2-KD mice during exercise despite normal extraction of glucose in skeletal muscle, led us to hypothesize that mitochondrial function is impaired in these mice
Summary
Animal Maintenance—All procedures were approved by the Vanderbilt University Animal Care and Use Committee. Two days prior to the exercise stress test, all mice were acclimatized to treadmill running by performing 10 min of exercise at a speed of 10 m1⁄7minϪ1 (0% incline). All mice performed a 10-min bout of exercise at their pre-determined experimental running speed (see below). The reaction was initiated by adding 30 g of protein to the assay buffer (25 mM potassium phosphate (pH 7.2), 5 mM MgCl, 2 mM KCN, 2.5 mg/ml bovine serum albumin (fraction V), 130 M NADH, 65 M decylubiquinone, 2 g/ml antimycin A) and monitored for 5 min. For the measurement of complex II ϩ III activity (succinate-cytochrome c oxidoreductase), 30 g of protein was added to 25 mM potassium phosphate (pH 7.2), 2 mM KCN, 20 mM succinate (pH 7.2), 2 g/l rotenone and incubated at 30 °C for 10 min.
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