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J Biol Chem. 2009 Sep 4; 284(36): 23925–23934.
Published online 2009 Jun 12. doi: 10.1074/jbc.M109.021048
PMCID: PMC2781986
PMID: 19525228

Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo*An external file that holds a picture, illustration, etc. Object name is sbox.jpg

Robert S. Lee-Young,‡,1 Susan R. Griffee,‡ Sara E. Lynes,‡ Deanna P. Bracy,‡ Julio E. Ayala,‡§ Owen P. McGuinness,‡§ and David H. Wasserman‡§
Author information Article notes Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

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 AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) 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 α2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ 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.

The ubiquitously expressed serine/threonine AMP-activated protein kinase (AMPK)2 is an αβγ heterotrimer postulated to play a key role in the response to energetic stress (1, 2), because of its sensitivity to increased cellular AMP levels (3). Pharmacological activation of AMPK (primarily via the AMP analogue ZMP) increases catabolic processes such as GLUT4 translocation (4, 5), glucose uptake (6, 7), long chain fatty acid (LCFA) uptake (8), and substrate oxidation (6). Concomitantly, pharmacological activation of AMPK inhibits anabolic processes, and in skeletal muscle genetic reduction of the catalytic AMPKα2 subunit eliminates these pharmacological effects (9–12). Thus, AMPK has been proposed to act as a metabolic master switch (2, 13, 14). Physiologically, exercise at intensities sufficient to increase free cytosolic AMP (AMPfree) levels is a potent stimulus of AMPK, preferentially activating AMPKα2 in skeletal muscle (15–17). The metabolic profile of skeletal muscle during moderate to high intensity exercise is remarkably similar to skeletal muscle in which AMPK has been pharmacologically activated (i.e. increases in catabolic processes). This is consistent with the hypothesis that AMPK activation is required for the metabolic response to increased cellular stress. Given this, it is surprising that the direct role(s) of skeletal muscle AMPK during exercise under physiological in vivo conditions is unknown.

A number of studies have tried to attribute causality to the AMPK and metabolic responses to exercise using transgenic models. In mouse models in which AMPKα2 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). Furthermore, ex vivo skeletal muscle LCFA uptake and oxidation in response to contraction appears to be AMPK-independent (20, 21). A key limitation of these studies is that the experimental models were not physiological. Under in vivo conditions, mice expressing a kinase-dead (18) or inactive (22) AMPKα2 subunit in cardiac and skeletal muscle have impaired voluntary and maximal physical activity, respectively, indicative of a physiological role for AMPK during exercise. In this context, obese non-diabetic and diabetic individuals have impaired skeletal muscle AMPK activation during moderate intensity exercise (23) as well as during the post-exercise period (24), yet the contribution of this impairment to the disease state is unclear. Thus, in vivo studies are essential to define the role of AMPK in skeletal muscle during exercise.

Physical exercise of a moderate intensity is an effective adjunct treatment for chronic metabolic diseases such as obesity and type 2 diabetes (25). Given the importance of elucidating the molecular mechanism(s) regulating skeletal muscle substrate metabolism during exercise and the putative role of AMPK as a critical mediator in this process, we tested the hypothesis that AMPKα2 is functionally linked to substrate metabolism in vivo.

EXPERIMENTAL PROCEDURES

Animal Maintenance

All procedures were approved by the Vanderbilt University Animal Care and Use Committee. Male and female C57BL/6J mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in cardiac and skeletal muscle (18) and wild-type (WT) littermate mice were studied. Twenty one days after birth, littermates were separated by gender, maintained in microisolator cages, fed a standard chow diet (5.5% fat by weight; 5001 Laboratory Rodent Diet, Purina), and had access to water ad libitum. All mice were studied at 16 weeks of age.

Exercise Stress Test

Peak oxygen consumption (V̇O2peak) was assessed using an exercise stress test protocol. 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 m·min−1 (0% incline). To determine V̇O2peak, mice were placed in an enclosed single lane treadmill connected to Oxymax oxygen (O2) and carbon dioxide (CO2) sensors (Columbus Instruments, Columbus, OH). Following a 30-min basal period, mice commenced running at 10 m·min−1 on a 0% incline. Running speed was increased by 4 m·min−1 every 3 min until mice reached exhaustion, defined as the time point whereby mice remained at the back of the treadmill on a shock grid for >5 s. O2 consumption and CO2 production were assessed at 30-s intervals throughout the basal and exercise periods. Basal values are representative of the final 10 min of the basal period. Prior to the V̇O2peak test, body weight was measured, and body composition was assessed using an mq10 NMR analyzer (Bruker Optics, The Woodlands, TX). Given that changes in whole body V̇O2 during exercise closely reflect changes occurring within exercising muscle (26), all oxygen consumption measurements were expressed per kg of lean body mass (kgLBM).

Metabolic Experiments

Following the exercise stress test, surgical procedures were performed as described previously (27) to catheterize the left common carotid artery and right jugular vein for sampling and infusions, respectively. The catheters were exteriorized, sealed with stainless steel plugs, and kept patent with saline containing 200 units·ml−1 heparin and 5 mg·ml−1 ampicillin. Mice were housed individually post-surgery, and body weight was recorded daily. Five days following surgery, all mice performed a 10-min bout of exercise at their pre-determined experimental running speed (see below). Experiments were performed 2 days later.

Approximately 1 h prior to the experiment, Micro-Renathane tubing was connected to the exteriorized catheters, and all mice were placed in the enclosed treadmill to acclimate to the environment. At t = 0 min, a base-line arterial blood sample was taken for the measurement of arterial glucose, plasma insulin, plasma nonesterified fatty acids (NEFA), plasma lactate, and hematocrit. Mice then remained sedentary or performed a single bout of exercise. Sedentary mice were allowed to move freely in the stationary treadmill for 30 min. Mice that exercised were divided into three groups as follows: 1) α2-KD mice performed a maximum of 30 min of treadmill exercise at 70% of their maximum running speed; 2) WT mice ran at the same absolute running speed as α2-KD mice; 3) WT mice ran at the same relative intensity as α2-KD mice. Running time was matched between groups.

In all mice, a bolus containing 13 μCi of 2-[14C]deoxyglucose (2-[14C]DG) and 26 μCi of [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) was injected into the jugular vein at t = 5 min to provide an index of tissue-specific glucose and LCFA uptake and clearance, respectively. At t = 7, 10, 15, and 20 min, arterial blood was sampled to determine blood glucose, plasma NEFA, plasma lactate, and plasma 2-[14C]DG and 3H-R-BrP. Hematocrit was measured at t = 20 min, and at t = 30 min or exhaustion, arterial blood was taken for the measurement of blood glucose, plasma insulin, plasma NEFA, plasma lactate, plasma 2-[14C]DG, and 3H-R-BrP. Following the final arterial blood sample, 50 μl of yellow DYE-TRAK® microspheres (15 μm; Triton Technology Inc., San Diego) were injected into the carotid artery, followed by a small flush of saline, to assess the percentage of cardiac output to gastrocnemius (%QG) and the left and right kidney. Mice were then anesthetized with an arterial infusion of sodium pentobarbital (3 mg). The soleus, right gastrocnemius, superficial vastus lateralis (SVL), heart, and brain were rapidly excised, frozen in liquid nitrogen, and stored at −70 °C. The left gastrocnemius and left and right kidney were placed into 15-ml polypropylene tubes and stored at 4 °C prior to microsphere analysis.

Echocardiography

Transthoracic echocardiograms were performed as described previously (28). Mice were acclimated to the procedure over 3 days. Immediately following treadmill exercise, two-dimensional targeted M-mode echocardiographic images were obtained at the level of the papillary muscles from the parasternal short axis view and recorded at a speed of 150 cm/s for the measurement of heart rate. Echocardiograms were completed within 72 ± 13 s after exercise. Left ventricular wall thickness, end diastolic measurements, and left ventricular end systolic dimensions were determined as described previously (28) and are the average of three to five consecutive selected sinus beats using the leading edge technique. Heart rate was determined from the cardiac cycles recorded on the M-mode tracing.

Plasma and Tissue Radioactivity

Plasma 2-[14C]DG radioactivity was assessed by liquid scintillation counting following deproteinization with 0.3 n Ba(OH)2 and 0.3 n ZnSO4 as described previously (29). Plasma 3H-R-BrP radioactivity was determined directly from the plasma via liquid scintillation counting. Tissue 2-[14C]DG and 3H-R-BrP were determined using a modified method of Folch et al. (30). Chloroform:methanol (2:1) was added to a portion of tissue that had been crushed in liquid nitrogen using a mortar and pestle, homogenized on ice, and stored at 4 °C for 60 min. KCl (0.1 m) was then added to the homogenate, and samples were centrifuged at 3500 × g for 15 min. The upper aqueous phase (containing 2-[14C]DG) was used to determine 2-[14C]DG -P as described previously (29). A portion of the lower lipid phase (containing 3H-R-BrP) was used to determine tissue 3H-R-BrP content (31).

Plasma Hormones and Metabolites

Immunoreactive plasma insulin was assayed with a double antibody method (32), and plasma NEFA were measured spectrophotometrically using an enzymatic colorimetric assay (NEFA C kit, Wako Chemicals Inc.). Plasma lactate was determined enzymatically (33), with lithium l-lactate (Sigma) used as the standard. Arterial glucose levels were determined directly from ∼5 μl of arterial blood samples using an ACCU-CHEK® Advantage monitor (Roche Diagnostics).

Muscle Metabolites

For muscle glycogen determination, 2 m HCl was added to a portion (∼10 mg) of crushed tissue samples, which were then incubated at 100 °C for 2 h and neutralized with 0.667 m NaOH. Glucose units were determined using an enzymatic fluorometric method (33). Muscle lactate, PCr, Cr, and ATP were analyzed from ∼20 mg of crushed tissue using enzymatic fluorometric techniques (33). ADPfree and AMPfree were calculated as described previously (34).

Microsphere Isolation

Tissues were digested overnight in 1 m KOH at 60 °C. Following sonication with Triton X-100, microspheres were suspended in ethanol containing 0.2% (v/v) HCl, followed by ethanol. The microsphere:ethanol solution was evaporated at room temperature, and 200 μl of N,N-dimethylformamide (Sigma) was added to elute the fluorescent dye from the microspheres. The absorbance of the N,N-dimethylformamide solution was determined at 450 nm.

Immunoblotting

Muscle samples were homogenized in lysis buffer (50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml trypsin inhibitor, 5 μl/ml protease inhibitor mixture, 50 mm NaF, and 5 mm sodium pyrophosphate). Samples were centrifuged at 10,000 × g, and protein content in the supernatant was determined using the Bradford method. Protein expression of AMPKα1 and -α2, acetyl-CoA carboxylase-β, neuronal (n) nitric-oxide synthase (NOS), and endothelial (e) NOS was determined from 75 μg of whole cell lysate. Inducible NOS was immunoprecipitated using 200 μg of protein in conjunction with immobilized Recomb protein A beads (Pierce) and an anti-inducible NOS mouse monoclonal antibody (BD Biosciences). Proteins were separated using NuPAGE 4–12% BisTris gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. Blots were probed with anti-AMPKα1 rabbit monoclonal antibody (1:500; Abcam, Cambridge, MA), anti-AMPKα2 goat polyclonal antibody (1:100; Santa Cruz Biotechnology), anti-nNOS mouse monoclonal antibody (1:500; BD Biosciences), anti-eNOS rabbit polyclonal antibody (1:100; Abcam, MA), and anti-inducible NOS mouse monoclonal antibody (1:100; BD Biosciences). Antibody binding was detected with either IRDyeTM 800-conjugated anti-rabbit IgG (1:10,000), IRDyeTM 700-conjugated anti-mouse IgG (1:10,000), or IRDyeTM 800-conjugated anti-goat IgG secondary antibodies (Rockland Immunochemicals, Inc., Gilbertsville, PA). Acetyl-CoA carboxylase-β protein expression was detected using IRDyeTM 800-labeled streptavidin (1:5,000; Rockland).

AMPK and NOS Activity Assays

AMPKα2 and -α1 were sequentially immunoprecipitated using 200 μg of protein, 2 μg of a rabbit AMPKα2 polyclonal antibody (Abcam), 2 μl of a rabbit AMPKα1 monoclonal antibody (Abcam), and immobilized Recomb protein A beads (Pierce). AMPK activity in the immune complexes was measured for 24 min at 30 °C (within the pre-determined linear range) in the presence of 200 μm AMP and calculated as picomoles of phosphate incorporated into the SAMS peptide (100 μm; GenWay Biotech) per min per mg of protein subjected to immunoprecipitation.

NOS activity was measured on gastrocnemius and SVL muscle. Samples were homogenized in lysis buffer, and 5 μl of sample (∼70 μg of protein) was added to pre-heated assay buffer (1.15 mm NADPH, 4 μm BH4, 100 nm calmodulin, 0.7 mm CaCl, 0.63 μm FAD, 3 μm l-[3H]arginine). The assay was performed for 7 min at 37 °C (within the linear range), and NOS activity was measured with or without the NOS inhibitor Nω-nitro-l-arginine methyl ester (1 mm). NOS activity is the difference between samples incubated with or without Nω-nitro-l-arginine methyl ester and was calculated as picomoles of l-[3H]arginine converted to picomoles of l-[3H]citrulline per min per mg of protein.

OXPHOS Activity Assays

Post-600 × g supernatants of gastrocnemius muscle were prepared as described previously (35). Briefly, frozen samples were homogenized in 120 mm KCl, 20 mm HEPES (pH 7.4), 2 mm MgCl, 1 mm EGTA, and 5 mg/ml bovine serum albumin and centrifuged twice at 600 × g for 10 min at 4 °C. The second supernatant was stored in 2 μg/μl aliquots at −70 °C. All assays were performed at 30 °C in a final volume of 1 ml using a SpectraMax Plus384 spectrophotometer (Molecular Devices). Prior to measurement of complex I, I + III, II, and II + III activity, samples were diluted 1:1 in hypotonic media (final concentration of 25 mm potassium phosphate (pH 7.2), 5 mm MgCl) and freeze-thawed three times.

Complex I activity (NADH:ubiquinone oxidoreductase; EC 1.6.5.3) was measured by following the decrease in absorbance due to the oxidation of NADH at 340 nm, with 425 nm as the reference wavelength (ϵ = 6.81 mm−1·cm−1) (35). The reaction was initiated by adding 30 μg 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 μm NADH, 65 μm decylubiquinone, 2 μg/ml antimycin A) and monitored for 5 min. Rotenone (2 μg/ml) was added, and the reaction was monitored for 3 min. Complex I activity is the difference between total enzymatic rates and rates obtained in the presence of rotenone. Complex I + III (NADH-cytochrome c oxidoreductase) activity was determined as described previously (36) with minor modifications. The reaction was initiated by adding 30 μg of protein to the assay buffer (50 mm potassium phosphate (pH 7.2), 80 μm cytochrome c (bovine heart), 130 μm NADH, 2 mm KCN, 5 mm MgCl). The increase in absorbance due to the reduction of ferricytochrome c (ϵ = 19 mm−1·cm−1) was monitored for 3 min at 550 nm with 580 nm as the reference wavelength. Rotenone (2 μg/ml) was added, and the reaction was monitored for a further 3 min. Complex I + III activity is the rotenone-sensitive rate.

Complex II activity (succinate:ubiquinone oxidoreductase; EC 1.3.5.1) was measured by following the reduction of 2,6-dichlorophenolindophenol at 600 nm with 750 nm as the reference wavelength (ϵ = 19.1 mm−1·cm−1) (35). Samples (30 μg) were incubated in 25 mm potassium phosphate (pH 7.2), 5 mm MgCl, and 20 mm succinate (pH 7.2) for 10 min at 30 °C. Antimycin A (2 μg/ml), rotenone (2 μg/ml), KCN (2 mm), and 2,6-dichlorophenolindophenol (50 μm) were added, and the reaction was monitored for 3 min. Decylubiquinone (65 μm) was added, and the reaction was monitored for a further 3 min. For the measurement of complex II + III activity (succinate-cytochrome c oxidoreductase), 30 μg of protein was added to 25 mm potassium phosphate (pH 7.2), 2 mm KCN, 20 mm succinate (pH 7.2), 2 μg/μl rotenone and incubated at 30 °C for 10 min. Ferricytochrome c was added (37.5 μm), and the increase in absorbance due to the reduction of ferricytochrome c was measured for 3 min at 550 nm with 580 nm as the reference wavelength.

Complex IV activity (cytochrome c oxidase; EC 1.9.3.1) was measured by following the decrease in absorbance at 550 nm due to the oxidation of ferrocytochrome c, with 580 nm as the reference wavelength (ϵ = 19.1 mm−1·cm−1) (35). Samples (10 μg) were added to 20 mm potassium phosphate, 15 μm ferrocytochrome c, and 450 μm n-dodecyl β-d-maltoside, and the reaction was monitored for 30 s. Complex IV activity was calculated from the initial rate. Ferrocytochrome c was prepared by adding 5 μm dithiothreitol to 200 μm ferricytochrome c. After 20 min, the 550 nm/565 nm ratio was determined, and ferricytochrome c was considered reduced if the ratio was between 10 and 20. Citrate synthase was measured on 10 μg of sample as described by Barrientos (37).

Calculations

The tissue-specific clearance of 2-[14C]DG and 3H-R-BrP (Kg and Kf, respectively) and the metabolic index for glucose and LCFA (Rg and Rf) were calculated as described previously (38). Kg and Kf are used as concentration-independent indices of muscle glucose and LCFA uptake, respectively. Rg and Rf are concentration-dependent indices of muscle glucose and LCFA uptake, respectively.

Percent cardiac output was calculated from fluorescent intensity as described previously (39) and is expressed as percent cardiac output to the tissue (%QT), where %QT = (fT/fRef)·(tissueaverage/tissuemouse). fT and ƒRef are the fluorescent intensity of the tissue and reference sample, respectively. Adequacy of microsphere mixing was assumed if %Q to the left and right kidney was within 10%. Of the 43 mice infused with microspheres, 34 met the inclusion criteria for analysis.

The amount of 2-[14C]DG-P present in the gastrocnemius muscle as well as the amount of microspheres trapped within the gastrocnemius muscle were used to determine the glucose tissue extraction index (TEI). The glucose TEI was calculated by expressing the percentage of 2-[14C]DG-P (expressed relative to the amount infused) relative to the percentage of microspheres (expressed relative to the amount infused). For the echocardiography experiments, an index linearly related to cardiac output was calculated as heart rate × (diastolic left ventricular internal dimension3 − systolic left ventricular internal dimension3) (28).

Statistical Analyses

Data are means ± S.E. Statistical analysis was performed using a Student's t test, one-way analysis of variance (ANOVA), one-way repeated measures ANOVA, or two-way repeated measures ANOVA where appropriate with the statistical software package SigmaStat. If the ANOVA was significant (p < 0.05), specific differences were located using Fisher's least significant difference test.

RESULTS

Exercise Capacity and Oxygen Consumption in Vivo Are Impaired in α2-KD mice during an Exercise Stress Test

At 16 weeks of age no significant differences were observed between α2-KD mice and WT littermates with respect to body weight (24 ± 2 versus 25 ± 1 g for WT and α2-KD, respectively), muscle mass (77 ± 1 versus 78 ± 2% body weight), or fat mass (8.5 ± 1.4 versus 9.4 ± 0.4% body weight). Basal V̇O2 was similar between genotypes (78 ± 5 versus 79 ± 6 ml·kgLBM−1·min−1 for WT and α2-KD, respectively) as was the respiratory exchange ratio (0.77 ± 0.02 versus 0.77 ± 0.01). During an exercise stress test, α2-KD mice displayed marked exercise intolerance as seen by impairments in maximum running speed (38 ± 1 versus 21 ± 1 m·min−1 for WT and α2-KD, respectively; p < 0.001) and running time (23 ± 1 versus 10 ± 1 min; p < 0.001). V̇O2 during the stress test increased at a similar rate in WT and α2-KD mice (Fig. 1A); however, V̇O2peak was reduced in α2-KD mice (142 ± 2 versus 113 ± 4 ml·kgLBM−1·min−1; p < 0.001). As a result, α2-KD mice were exercising at a greater percentage of V̇O2peak compared with WT mice at any given absolute work rate (supplemental Table S1). Respiratory exchange ratio was similar between genotypes at exhaustion (0.89 ± 0.01 versus 0.90 ± 0.03). At a V̇O2 of ∼90 ml·kgLBM−1·min−1, V̇CO2 increased disproportionately compared with V̇O2 in WT mice (Fig. 1B), reflecting a change in either substrates utilized or acidosis. This effect was not apparent in α2-KD mice (Fig. 1C).

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Oxygen consumption is impaired in 16-week-old chow-fed C57BL/6J mice expressing a kinase-dead form of AMP-activated protein kinase α2 (α2-KD) in cardiac and skeletal muscle. Compared with WT littermates, the increase in oxygen consumption (ΔV̇O2) during an exercise stress test is attenuated in α2-KD mice (A). B and C, carbon dioxide production (V̇CO2) during an exercise stress test was plotted against V̇O2 for WT and α2-KD mice, respectively. Note the change of slope of V̇CO2 versus V̇O2 in WT mice (denoted by the arrow) that is not present in α2-KD mice. Data are mean ± S.E. for n = 8–9. kgLBM indicates kilograms of lean body mass.

Acute Exercise Experiment, Controlling for Relative and Absolute Exercise Intensity

To examine the role of AMPKα2 in the regulation of skeletal muscle metabolic flux in vivo, α2-KD mice performed a single bout of treadmill exercise at 70% of their maximum running speed (α2-KD70%). Because of the difference in maximum running speed between the genotypes, WT mice that exercised at the same absolute speed as α2-KD70% did so at ∼45% of their maximum running speed (WT45%; supplemental Table S2). To best equate results to α2-KD70%, a second group of WT mice was exercised at 70% of their maximum running speed (WT70%; supplemental Table S2). As demonstrated in the results that follow, controlling for absolute and relative exercise intensity is essential for interpretation of the physiological and metabolic responses to exercise in vivo.

AMPKα Protein Expression and AMPK Activity Is Impaired in Skeletal Muscle of α2-KD Mice

Similar to other muscle groups (11), expression of the α2-KD subunit in gastrocnemius muscle was increased relative to native AMPKα2 (98 ± 8% higher in α2-KD compared with WT, p < 0.01; supplemental Fig. S1A). A concomitant decrease in AMPKα1 expression was observed in the gastrocnemius of α2-KD mice (51 ± 11% lower in α2-KD compared with WT, p < 0.02; supplemental Fig. S1A). Similar findings for AMPKα2 and α1 expression were observed in SVL muscle (data not shown). In gastrocnemius muscle of WT mice, AMPKα2 (supplemental Fig. S1B) and AMPKα1 activities (supplemental Fig. S1C) increased in an intensity-dependent manner. AMPKα2 and -α1 activities were barely detectable in the gastrocnemius of α2-KD mice under sedentary conditions and did not change in response to exercise. Gastrocnemius acetyl-CoA carboxylase-β Ser221 phosphorylation was similar between genotypes at rest and increased to a similar extent in all groups in response to exercise (supplemental Fig. S1D).

Skeletal Muscle ATP Concentrations Decrease in α2-KD Mice during Exercise in Vivo

In response to exercise, no significant changes in ATP were observed in the gastrocnemius of WT45% or WT70% (Table 1). In contrast, exercise significantly decreased gastrocnemius ATP levels in α2-KD70%. Lactate and creatine (Cr) significantly increased, whereas phosphocreatine (PCr), PCr:(PCr + Cr), and glycogen significantly decreased during exercise in all groups (Table 1). In α2-KD70% and WT70%, ADPfree, AMPfree, and AMPfree:ATP all increased in response to exercise (Table 1). The similar increase in AMPfree and AMPfree:ATP observed between α2-KD70% and WT70% shows that, by this criteria, cellular stress was equally elevated in these groups compared with WT45%. This finding emphasizes the need to exercise mice at the same relative work intensity to obtain comparable energetic responses in vivo.

TABLE 1

Measured and calculated metabolites (normalized to total creatine levels) and glycogen at rest and immediately following exercise in gastrocnemius muscle of 16-week-old chow-fed C57BL/6J mice expressing WT or kinase-dead form of AMP-activated protein kinase α2 (α2-KD) in cardiac and skeletal muscle

Data are mean ± S.E. for n = 5–7 per group.

Metabolite
Sedentary
Exercise
WTα2-KDWT45%WT70%α2-KD70%
ATP (μmol·100 g−1)29.1 ± 2.230.5 ± 1.625.7 ± 1.125.4 ± 1.219.7 ± 2.7a
Lactate (μmol·100 g−1)12.3 ± 2.212.3 ± 1.757.7 ± 14.1a54.0 ± 12.6a78.4 ± 15.5a
PCr (μmol·100 g−1)62.4 ± 3.157.8 ± 3.328.5 ± 8.3a29.5 ± 8.5a16.1 ± 5.2a
Cr (μmol·100 g−1)37.6 ± 3.142.2 ± 3.371.5 ± 8.3a70.5 ± 8.5a83.9 ± 5.2a
PCr:(PCr + Cr)0.60 ± 0.030.58 ± 0.030.28 ± 0.08a0.29 ± 0.08a0.12 ± 0.03a
ADPfree (nmol·100 g−1)154 ± 19178 ± 22255 ± 57a372 ± 90a451 ± 117a
AMPfree (nmol·100 g−1)0.9 ± 0.21.2 ± 0.23.2 ± 1.37.8 ± 3.4a7.5 ± 2.3a
AMPfree:ATP0.05 ± 0.010.03 ± 0.000.13 ± 0.060.33 ± 0.15a0.42 ± 0.13a
Glycogen (μmol·100 g−1)901 ± 174630 ± 101390 ± 106a345 ± 62a162 ± 34a

a p < 0.05 versus corresponding basal.

Arterial Metabolites and Hormones Are Altered in α2-KD Mice at Rest and during Steady State Exercise in Vivo

An increase in exercise intensity resulted in significantly lower arterial glucose levels in WT mice (Fig. 2A). Compared with WT70%, arterial glucose levels during exercise were significantly greater in α2-KD70%. Arterial NEFAs (Fig. 2B) and insulin (Fig. 2C) decreased to similar concentrations in all groups during exercise. Although no differences in basal insulin levels were observed between individual groups, basal insulin levels were greater in α2-KD70% compared with the combined average of all WT mice (98 ± 6 versus 71 ± 7 pm, p < 0.05). Arterial lactate increased over time in all exercise groups (Fig. 2D), and a significant group effect was observed with α2-KD70% > WT70% > WT45% (p < 0.01).

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Arterial blood glucose (A), plasma NEFAs (B), plasma insulin (C), and plasma lactate (D) at rest and during exercise in 16-week-old chow-fed C57BL/6J mice expressing a WT or kinase-dead form of AMP-activated protein kinase α2 (α2-KD) in cardiac and skeletal muscle. Following a 1-h fast, chronically catheterized mice performed a maximum of 30 min of running on a motorized treadmill, and arterial blood was sampled at times shown. Data are mean ± S.E. for n = 7–9 per group. †, p < 0.05 versus corresponding basal value; **, p < 0.01 versus α2-KD70%; f, main effect for time, p < 0.05; § main effect for group, p < 0.01.

Indices of Glucose Uptake, but Not LCFA Uptake, Are Impaired in Skeletal Muscle of α2-KD Mice during Exercise in Vivo

In WT mice, an increase in exercise intensity increased the plasma disappearance of 2-[14C]DG at 7 and 10 min (Fig. 3A). Gastrocnemius Kg (Fig. 3B) and Rg (Fig. 3C) also increased in WT70% compared with WT45%. At the same relative exercise intensity, the disappearance of plasma 2-[14C]DG was attenuated at 7 min in α2-KD70% mice when compared with WT70%. In α2-KD70% mice, gastrocnemius Kg was impaired by ∼60% when compared with WT70% mice (Fig. 3B). Gastrocnemius Rg during exercise was also impaired ∼35% in α2-KD70% mice when compared with WT70% (Fig. 3C).

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Plasma 2-[14C]DG clearance (A), gastrocnemius glucose clearance (Kg; B), glucose uptake (Rg; C), percent cardiac output to gastrocnemius (%QG; D), and the glucose TEI (E) in 16-week-old chow-fed C57BL/6J mice expressing a WT or kinase-dead (KD) form of AMP-activated protein kinase α2 in cardiac and skeletal muscle. Following a 1-h fast, mice were given a bolus injection of 13 μCi of 2-[14C]DG, and Rg and Kg values were calculated as described (see “Experimental Procedures”). Following a final blood sample, fluorescent microspheres were injected into the carotid artery to determine %QG (see “Experimental Procedures”). Data are mean ± S.E. for n = 8–9 per groups except for D and E where n = 5–7 per group. *, p < 0.05 versus corresponding WT; **, p < 0.05 versus WT70%; ***, p < 0.05 versus α2-KD70%; †, p < 0.05 versus corresponding sedentary group.

An increase in exercise intensity tended to increase Kg in the soleus of WT mice (p = 0.07 for WT70% versus WT45%; supplemental Fig. S2A), whereas Kg in WT70% was significantly greater than WT45% in SVL (supplemental Fig. S2B). These results paralleled findings observed for Rg in soleus (supplemental Fig. S2C) and SVL (supplemental Fig. S2D). As with the gastrocnemius, Kg in soleus and SVL was impaired ∼30% in α2-KD70% mice when compared with WT70%; however, soleus and SVL Rg was similar between α2-KD70% and WT70%.

Taken together, these findings show that glucose concentration-dependent (Rg) and -independent (Kg) indices of MGU are impaired in α2-KD mice during exercise in vivo compared with WT mice exercising at the same relative intensity. The finding that MGU was greater in WT70% compared with WT45% shows for the first time that the 2-[14C]DG method (38) can be used to determine the effect of different exercise intensities on multiple muscle groups in vivo.

Indices of LCFA clearance (Kf) and uptake (Rf) are shown in supplemental Fig. S3. Kf increased to similar rates during exercise in soleus, gastrocnemius, and SVL of α2-KD70% and WT70%. In WT45% Kf responses were generally reduced. Rf significantly increased in soleus, gastrocnemius, and SVL of α2-KD70%. In WT70%, Rf significantly increased in soleus and gastrocnemius, whereas Rf was elevated in gastrocnemius of WT45%. Given that Kf and Rf increased normally in response to exercise in α2-KD mice, it can be concluded that AMPKα2 is not essential for skeletal muscle LCFA uptake during exercise in vivo. This is in agreement with previous studies performed ex vivo (20, 21).

Percent Cardiac Output to Skeletal Muscle Is Altered in α2-KD Mice at Rest and during Exercise in Vivo

Under basal conditions %QG was ∼2.5-fold greater in α2-KD mice compared with WT mice (Fig. 3D). Exercise increased %QG in WT45% (∼4.5-fold) and WT70% (∼4-fold). Exercise did not alter %QG in α2-KD70%. The glucose TEI did not differ between α2-KD and WT mice at rest (Fig. 3E). Exercise increased the glucose TEI to a similar extent in α2-KD70% and WT70%, demonstrating that the impairment in MGU seen in the gastrocnemius of α2-KD70% compared with WT70% during exercise was likely due to reduced substrate delivery (i.e. %QG). The TEI did not increase in WT45%, demonstrating that in WT mice the extraction of glucose by skeletal muscle is accelerated as exercise intensity increases.

Cardiac Fuel Uptake and Function during Exercise in Vivo Are Not Impaired in α2-KD Mice

Cardiac Kg was similar between genotypes at rest, and exercise did not significantly increase Kg in any group (supplemental Fig. S4A). Cardiac Rg was also similar between genotypes at rest, and exercise significantly increased cardiac Rg in α2-KD70% and WT70% (supplemental Fig. S4B). Cardiac Rg did not increase during exercise in WT45% and was significantly less than cardiac Rg in α2-KD70% and WT70%. No significant differences were observed with respect to Kf or Rf in cardiac muscle between any of the three groups (data not shown). Heart rate (683 ± 12 versus 669 ± 9 versus 650 ± 5 beats·min−1 for WT45%, WT70%, and α2-KD70%, respectively) and cardiac output (14 ± 1 versus 14 ± 1 versus 15 ± 1 ml·min−1 for WT45%, WT70%, and α2-KD70%, respectively) were similar between groups in response to exercise. Thus, a kinase-dead AMPKα2 subunit in cardiac muscle does not adversely affect substrate uptake or cardiac function in response to exercise.

NOS Expression and Activity Are Reduced in Skeletal Muscle of α2-KD Mice

In skeletal muscle, AMPK has been shown to interact with the endothelial and neuronal isozymes of NOS (eNOS and nNOS, respectively) (8, 40). In gastrocnemius of α2-KD mice, expression of the skeletal muscle isoform of nNOS (nNOSμ) was reduced ∼35% compared with WT mice (Fig. 4A). Gastrocnemius eNOS expression was similar between genotypes (0.05 ± 0.01 versus 0.04 ± 0.00 arbitrary units for WT and α2-KD, respectively), whereas expression of inducible NOS was also similar (0.32 ± 0.05 versus 0.37 ± 0.02 arbitrary units for WT and α2-KD, respectively). nNOSμ expression was also impaired in SVL of α2-KD mice (0.77 ± 0.09 versus 0.50 ± 0.05 arbitrary units for WT and α2-KD, respectively, p < 0.02).

An external file that holds a picture, illustration, etc. Object name is zbc0370984990004.jpg

NOS protein expression (A) and NOS activity (B) in gastrocnemius muscle of 16-week-old chow-fed C57BL/6J mice expressing a WT or kinase-dead (KD) form of AMP-activated protein kinase α2 in cardiac and skeletal muscle. A, SDS-PAGE was performed on 75 μg of whole cell lysate from gastrocnemius muscle. B, NOS activity at rest and in response to exercise was determined in gastrocnemius muscle as described (see “Experimental Procedures”). Data are mean ± S.E. for n = 5 per group. *, p < 0.05 versus corresponding WT; **, p < 0.05 versus WT70%; †, p < 0.05 versus corresponding sedentary group.

In the basal state, total NOS activity was ∼35% lower in gastrocnemius of α2-KD mice (Fig. 4B). Exercise increased NOS activity in WT70% but not in either WT45% or α2-KD70%. Basal NOS activity was also impaired in SVL muscle of α2-KD mice (supplemental Fig. S5); however, exercise did not alter SVL NOS activity in any group, a finding that may be related to less recruitment of this muscle (i.e. attenuated Rg and Kg when compared with gastrocnemius muscle). Thus, AMPK is required for full expression of nNOSμ, as well as NOS activity at rest and in response to exercise. The observation that NOS activity increased in gastrocnemius of WT70% but not WT45% shows that NOS activity is sensitive to exercise intensity.

Activities of Specific Electron Transport Chain (ETC) Complexes Are Reduced in Skeletal Muscle of α2-KD Mice

The finding that exercise capacity, V̇O2peak, and ATP generation are impaired, although changes in arterial lactate levels are accelerated in α2-KD mice during exercise despite normal extraction of glucose in skeletal muscle, led us to hypothesize that mitochondrial function is impaired in these mice. Support for this hypothesis comes from the finding that a reduction in nNOSμ protein expression, such as seen in the present study, is associated with impaired activity of enzymes involved in skeletal muscle OXPHOS (41, 42). As shown in Table 2, complex I and complex IV activities of the ETC were significantly impaired in sedentary α2-KD mice when compared with WT mice, whereas no changes were observed for complex I + III, II, or II + III activities. Identical findings were observed if complex activities were normalized to citrate synthase levels, which did not differ between genotypes (50 ± 7 versus 52 ± 11 μmol·min−1·mg−1 for WT and α2-KD, respectively). Thus, the impairment in skeletal muscle ETC complexes in α2-KD mice was not because of a nonspecific reduction in mitochondrial content, a finding that is in agreement with previous observations demonstrating no alteration in mitochondrial density, DNA, and other markers of mitochondrial content and biogenesis in gastrocnemius muscle of untrained α2-KD mice (43).

TABLE 2

Electron transport chain complex activities in gastrocnemius muscle of 16-week-old chow-fed C57BL/6J mice expressing a WT or kinase-dead form of AMP-activated protein kinase α2 (α2-KD) in cardiac and skeletal muscle

Data are mean ± S.E. for n = 5–6 per group. Activities are expressed as nmol·min−1·mg−1.

ComplexEnzymeWTα2-KD
INADH:ubiquinone oxidoreductase74.9 ± 7.451.0 ± 5.9a
I + IIINADH:cytochrome c oxidoreductase11.1 ± 3.76.6 ± 2.6
IISuccinate:ubiquinone oxidoreductase10.7 ± 2.19.5 ± 2.7
II + IIISuccinate:cytochrome c oxidoreductase13.4 ± 3.08.0 ± 1.7
IVCytochrome c oxidoreductase144.7 ± 15.272.3 ± 9.4a

a p < 0.05 versus corresponding WT.

DISCUSSION

This study supports for the first time in vivo the hypothesis that AMPK is a critical mediator of the metabolic response to exercise. We demonstrate that AMPK regulates skeletal muscle metabolism in vivo at multiple levels, with the overall result being that a defect in AMPKα2 subunit activity in skeletal muscle grossly impairs exercise tolerance. Without a functionally active AMPKα2 subunit, glucose uptake during exercise in vivo is impaired in different skeletal muscle groups of α2-KD mice compared with WT littermate mice exercising at the same relative intensity. This may be due in part to impaired substrate delivery to exercising muscle (estimated via %QG) as the glucose TEI was not different between the two genotypes. Specific ETC complex activities in skeletal muscle were also impaired in α2-KD mice. Taken together with the findings of decreased skeletal muscle ATP concentrations, greater arterial lactate accumulation, and reductions in V̇O2peak during maximal exercise in α2-KD mice, our findings suggest that the exercise intolerance in α2-KD mice is the result of impaired energy-producing oxidative pathways (see Fig. 5).

An external file that holds a picture, illustration, etc. Object name is zbc0370984990005.jpg

Proposed model describing the role of skeletal muscle AMPKα2 during exercise in vivo. Our results show that skeletal MGU during exercise is dependent on AMPKα2 activation, as mice expressing α2-KD have impaired MGU when compared with WT mice at the same relative exercise intensity. The impaired MGU in α2-KD mice is at least partially because of reduced vasodilation, which arises from an inability of AMPK to activate NOS and thus stimulate NO production. The impairment in AMPKα2 activation and/or reductions in the skeletal muscle isoform of neuronal NOS (nNOSμ) also attenuate mitochondrial function. This reduces mitochondrial ATP generation and diverts glucose toward anaerobic ATP generation, resulting in elevated plasma lactate levels. The whole body phenotype of these impairments is a reduction in exercise tolerance. G-6-P, glucose 6-phosphate.

The novel finding that complex I and complex IV activities of the ETC were impaired in the gastrocnemius of α2-KD mice reveals new insight regarding the role of AMPK in skeletal muscle, and it provides a mechanism that could account for or contribute to the exercise intolerance observed in the α2-KD mouse. Complex I and complex IV represent the proximal and distal ETC complexes, respectively, and thus play an integral role in OXPHOS and the generation of ATP. A deficiency in complex I activity will lead to excess levels of NADH and a lack of NAD+, resulting in impaired Krebs cycle function and elevated blood lactate (44), the latter being observed in α2-KD mice during exercise in this study. A deficiency in complex IV activity would impair the proton gradient required for subsequent ATP synthesis (45), explaining the accelerated net ATP degradation observed in skeletal muscle of α2-KD mice during exercise in vivo. Importantly, the changes in complex I and complex IV activities in α2-KD mice occurred despite similar levels of citrate synthase activity when compared with WT mice. This agrees with previous findings showing that mitochondrial density, mitochondrial DNA, cytochrome c protein expression, δ-aminolevulinate synthase mRNA expression, and peroxisome proliferator-activated receptor γ coactivator-1α mRNA expression are similar in gastrocnemius muscle of untrained α2-KD and WT mice (43). Thus, a functionally inactive AMPKα2 subunit is sufficient to impair mitochondrial function, without adversely altering markers of muscle mitochondrial content.

Although OXPHOS capacity was impaired in skeletal muscle of α2-KD mice, it is unclear whether the α2-KD subunit per se was directly responsible for this phenomenon. A novel finding with important implications was that nNOSμ protein expression was impaired in skeletal muscle of α2-KD mice. This finding is supported by the close association between AMPKα2 and nNOSμ (40). A decrease in nNOSμ protein expression has been associated with impairments in OXPHOS. Indeed, in skeletal muscle of patients with amyotrophic lateral sclerosis, reduced nNOSμ expression is highly associated with impaired ETC complex activities (42). Similarly, in skeletal muscle of nnosμ−/− mice, ETC complex activities are reduced (41). Thus, the impairments in OXPHOS within skeletal muscle of α2-KD mice may be due to a direct impairment of AMPK or indirect effects mediated by reductions in nNOSμ protein expression.

The reduced nNOSμ expression may have also caused an impairment in muscle blood flow, as %QG did not increase in response to exercise in α2-KD mice, whereas an ∼4-fold increase was observed in WT70% and WT45%. It has been shown that vasodilation in response to mild exercise is significantly impaired in animal models where nNOSμ is partially impaired or ablated in skeletal muscle (46). Likewise, Lau et al. (47) have shown that ∼50% of contraction-induced arteriolar dilation in vitro is dependent on nNOSμ. Conversely, restoring nNOSμ at the sarcolemma of skeletal muscle significantly improves the exercise-induced increase in skeletal muscle perfusion (48). It is well known that contracting muscle releases nitric oxide (NO) (49, 50). Given that NOS activity in gastrocnemius of α2-KD mice was impaired in response to intense exercise, it is a plausible hypothesis that NO efflux from α2-KD mice was also impaired. NO is a potent stimulator of vasodilation (51), and as such impaired NOS activity during exercise may have also suppressed arteriolar relaxation in α2-KD mice. Aside from nNOSμ, it has been shown that the gastrocnemius of α2-KD mice contains significantly fewer capillaries compared with WT mice (52). Given that exercise normally causes a redistribution of blood flow toward contracting muscle (53), fewer capillaries in the gastrocnemius of α2-KD mice might have also resulted in less blood flow to this tissue during exercise.

We found for the first time that suppressed activation of AMPKα2 in skeletal muscle during exercise in vivo was associated with ∼60 and ∼35% reductions in concentration-independent and -dependent indices of MGU, respectively. These impairments became apparent when work intensity was normalized to the same relative work rate in WT and α2-KD mice. As mentioned above, a novel observation in α2-KD mice was that %QG did not increase in response to exercise. As a result, the impairment in MGU in α2-KD mice could at least partially be ascribed to a reduction in vascular glucose delivery to the contracting muscle. In line with this theory, we demonstrated that the glucose TEI in gastrocnemius of α2-KD mice was similar to WT mice exercising at the same relative intensity, suggesting that the muscle had adequate capacity to extract glucose from the blood.

A key aspect of this study was utilizing WT mice exercising at the same relative and absolute levels as α2-KD mice. To date, we are unaware of any study that has controlled for this variable when utilizing exercise as a means to amplify metabolic signals in rodents. Dzamko et al. (21) recently reported that V̇O2 and whole body substrate oxidation (assessed via indirect calorimetry) was similar between α2-KD and WT mice exercising at the same absolute running speed. This agrees with findings from this present study; however, the findings of Dzamko et al. (21) do not account for the difference in relative exercise intensity. Our exercise stress test demonstrated that at the same absolute running speed α2-KD mice were exercising at a greater percentage of V̇O2peak. This is evidenced by the elevated cellular stress in skeletal muscle of α2-KD mice (i.e. AMPfree and AMPfree:ATP) compared with WT mice at the same absolute running speed, observations also observed in AMPKα2−/− and WT mice exercising at the same absolute speed (54). Furthermore, in the present study Kg and Rg were elevated in SVL of α2-KD70% compared with WT45% demonstrating that this muscle group, comprised primarily of fast glycolytic fibers, was recruited to a greater extent in α2-KD mice. Based on these observations, we propose that the relative exercise intensity should be compared in rodent exercise studies, as is generally done in human exercise studies. Performing an exercise stress test in rodents provides valuable data pertaining to maximum running speed and running time and facilitates interpretation of subsequent studies examining physiological responses to an acute bout of exercise in vivo.

Aside from examining the role of AMPKα2 in skeletal muscle during exercise, we also addressed the role of AMPKα2 in cardiac muscle. Expression of the α2-KD transgene is driven by the muscle creatine kinase promoter (18), which is present in cardiac and skeletal muscle (55). The muscle creatine kinase promoter activity, and thus expression of the α2-KD transgene, is much lower in cardiac muscle (18). Nevertheless, cardiac function of α2-KD mice has been shown to be significantly impaired in metabolically challenged states such as ischemia (56), and it has been suggested that the exercise intolerance of α2-KD mice may be due to impairments in cardiac function as opposed to skeletal muscle defects (21, 57). In this study changes in cardiac glucose and LCFA uptake, heart rate, and cardiac output in α2-KD mice were similar to WT mice exercising at the same relative intensity. Thus, a functionally inactive AMPKα2 subunit in cardiac muscle does not appear to impair substrate uptake or cardiac function during physiological exercise conditions.

In conclusion, we show for the first time that exercise performed in vivo by α2-KD mice elicits a phenotype characterized by impaired V̇O2peak, exercise intolerance, enhanced ATP degradation in skeletal muscle, and lactic acidosis. At the same relative exercise intensity, MGU is impaired in α2-KD mice compared with WT mice. This is not because of attenuation in the fractional extraction of glucose by skeletal muscle but likely to impaired vascular glucose delivery to skeletal muscle. We also show that AMPK regulates nNOSμ protein expression and NOS activity, as well as mitochondrial function in skeletal muscle. Based on existing literature (46), it is likely that the effects of impaired AMPK activation on vascular and mitochondrial function are to an extent mediated by changes in nNOSμ. Thus, our findings demonstrate novel roles for AMPK in skeletal muscle and provide new insight into the role of AMPK during physiological exercise. Our findings have implications for chronic metabolic disease states such as obesity and type 2 diabetes, which are characterized by suppressed skeletal muscle AMPKα2 activity during exercise.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Prof. Morris Birnbaum (University of Pennsylvania) for kindly supplying the α2-KD mice used for breeding. We thank Drs. ZhiZhang Wang and Jeffrey Rottman of the Vanderbilt Mouse Metabolic Phenotyping Center, Cardiovascular Pathophysiology Core, for performing the echocardiography. We also thank Associate Professor Rodney Snow (Deakin University, Victoria, Australia) for helpful discussions regarding the metabolite analyses.

*This work was supported, in whole or in part, by National Institutes of Health Grants U24 DK-59637 and R01 DK-54902 (to D. H. W.).

An external file that holds a picture, illustration, etc. Object name is sbox.jpgThe on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S5.

2The abbreviations used are:

AMPK
AMP-activated protein kinase
%QG
percent cardiac output to gastrocnemius muscle
α2-KD
AMPK α2 kinase-dead subunit
ETC
electron transport chain
eNOS
endothelial NOS
LCFA
long-chain fatty acid
MGU
muscle glucose uptake
NEFA
nonesterified fatty acid
NO
nitric oxide
NOS
nitric-oxide synthase
nNOS
neuronal NOS
nNOSμ
muscle isoform of nNOS
OXPHOS
oxidative phosphorylation
TEI
tissue extraction index
BisTris
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
ANOVA
analysis of variance
Cr
creatine
PCr
phosphocreatine
SVL
superficial vastus lateralis
2-[14C]DG
2-[14C]deoxyglucose
and 3H-R-BrP
[9,10-3H]-(R)-2-bromopalmitate
WT
wild type
ZMP
5-aminoimidazole-4-carboxamide riboside monophosphate .

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