during low-intensity exercise

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1 Am J Physiol Endocrinol Metab 296: E47 E55, First published October 21, 2008; doi: /ajpendo AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise Shinji Miura, 1 Yuko Kai, 1 Yasutomi Kamei, 1,3 Clinton R. Bruce, 4 Naoto Kubota, 2,5 Mark A. Febbraio, 4 Takashi Kadowaki, 2,5 and Osamu Ezaki 1 1 Nutritional Science Program and 2 Clinical Nutrition Program, National Institute of Health and Nutrition; 3 Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 4 Cellular and Molecular Metabolism Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Australia; and 5 Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Submitted 13 August 2008; accepted in final form 11 October 2008 Miura S, Kai Y, Kamei Y, Bruce CR, Kubota N, Febbraio MA, Kadowaki T, Ezaki O. 2 -AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise. Am J Physiol Endocrinol Metab 296: E47 E55, First published October 21, 2008; doi: /ajpendo A single bout of exercise increases glucose uptake and fatty acid oxidation in skeletal muscle, with a corresponding activation of AMP-activated protein kinase (AMPK). While the exercise-induced increase in glucose uptake is partly due to activation of AMPK, it is unclear whether the increase of fatty acid oxidation is dependent on activation of AMPK. To examine this, transgenic mice were produced expressing a dominant-negative (DN) mutant of 1 -AMPK ( 1 -AMPK-DN) in skeletal muscle and subjected to treadmill running. 1 -AMPK-DN mice exhibited a 50% reduction in 1 -AMPK activity and almost complete loss of 2 -AMPK activity in skeletal muscle compared with wild-type littermates (WT). The fasting-induced decrease in respiratory quotient (RQ) ratio and reduced body weight were similar in both groups. In contrast with WT mice, 1 -AMPK-DN mice could not perform high-intensity (30 m/min) treadmill exercise, although their response to low-intensity (10 m/min) treadmill exercise was not compromised. Changes in oxygen consumption and the RQ ratio during sedentary and low-intensity exercise were not different between 1 -AMPK-DN and WT. Importantly, at low-intensity exercise, increased fatty acid oxidation in response to exercise in soleus (type I, slow twitch muscle) or extensor digitorum longus muscle (type II, fast twitch muscle) was not impaired in 1 -AMPK-DN mice, indicating that 1 -AMPK-DN mice utilize fatty acid in the same manner as WT mice during low-intensity exercise. These findings suggest that an increased 2 - AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise. adenosine 5 -monophosphate-activated protein kinase; fasting; respiratory quotient ratio; fatty acid oxidation; mitochondria; 5-aminoimidazole-4-carboxamide-1- -D-ribofuranoside AN ACUTE BOUT OF EXERCISE increases skeletal muscle glucose uptake by translocating the intracellular glucose transporter 4 (GLUT4) to the plasma membrane (10, 14) and increases fatty acid oxidation by stimulating carnitine palmitoyl transferase 1 (CPT1) activity, which limits fatty acid transport into mitochondria (33, 41). Both steps may be mediated by activation of AMP-activated protein kinase (AMPK), a sensor of fuel levels in skeletal muscle (15). AMPK is a heterotrimer composed of an ( 1 and 2 )-catalytic subunit and ( 1 and 2 )- and ( 1, 2, and 3 )-noncatalytic subunits. Of the 12 possible subunit Address for reprint requests and other correspondence: S. Miura, or O. Ezaki, Nutritional Science Program, National Institute of Health and Nutrition, , Toyama, Shinjuku-ku, Tokyo , Japan ( shinjim@nih. go.jp or ezaki@nih.go.jp). combinations, only 3 exist in human skeletal muscle, namely 1 / 2 / 1, 2 / 2 / 1, and 2 / 2 / 3 (4). Expression of 3 is predominately restricted to glycolytic skeletal muscle [type II, i.e., extensor digitorum longus (EDL); Ref. 24]. It is expressed at very low levels in oxidative muscles (type I, i.e., soleus). The roles of 1, 2, and 3 in skeletal muscles for glucose metabolism during muscle contraction (or exercise) have been extensively studied; however, the importance of AMPK activity in regulating fatty acid oxidation during exercise is unclear. Mice expressing a dominant-negative (DN) AMPK transgene in skeletal muscle and 1 - and 2 -knockout mice have been produced (13, 19, 20, 28, 37), and the role of AMPK on contraction-induced glucose uptake has been investigated. Mu et al. (28) reported that glucose transport activity in soleus and EDL from 2 -AMPK-DN mice after in situ electrical contraction was only 30% less than that in wild-type (WT) mice. Fujii et al. (13) produced 1 - and 2 -AMPK-DN transgenic mice and found that these transgenic mice showed the same phenotype in which 2 -activity in skeletal muscle was barely detectable and 1 activity was partially reduced. Contraction-stimulated glucose transport in isolated EDL, tibialis anterior, or gastrocnemius was normal in 2 -AMPK-DN transgenic mice (13). In addition, when force production during contraction ex vivo was matched between WT littermates and 2 -AMPK-DN mice, a similar increase in contraction-induced glucose transport was observed in isolated EDL from both groups of mice (13). In whole body 1 - and 2 -knockout mice, glucose transport activity in electrically stimulated (100-Hz, 0.2-ms impulse for 10 min), isolated soleus and EDL from either 1 - or 2 -AMPK knockout subgroups was not impaired. This suggests that the two -isoforms can compensate for each other in terms of contraction-induced glucose uptake or that neither -isoform is involved in contraction-induced glucose uptake (19). These studies used tetanic stimulation, a relatively strong electrical stimulation. However, increases in isoform specific AMPK activity differed by mode of electrical stimulation (36). In ex vivo experiments (in isolated EDL), electrical twitch contraction (1 and 2 Hz, 0.1 ms for 2 min) activated 1 -AMPK but not 2 -AMPK, whereas tetanic contraction (100-Hz, train duration 10 s, 10 min) activated both 1 -AMPK and 2 - AMPK activities (36). Both twitch and tetanic contractions could increase glucose uptake in EDL (36). Recently, it was reported (17) that increased glucose transport in response to The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /09 $8.00 Copyright 2009 the American Physiological Society E47

2 E48 electrical twitch contraction was not observed in 1 -AMPK knockout mice, suggesting that 1 -AMPK activity is required for stimulation of glucose uptake by twitch contraction. However, it is unknown whether low-intensity twitch contraction ex vivo is relevant to low-intensity exercise in vivo. We could find only one study (3) to examine the effects of exercise. After a swim bout, EDL muscles were excised from AMPK mutantoverexpression Tg-Prkag3 225Q (dominant-positive mutation) mice, AMPK 3 -knockout mice, and WT mice and incubated for 20 min to determine the rate of glucose uptake after exercise (3). Swimming increased glucose uptake to an equal extent in all genotypes. These findings strongly suggest that additional pathways mediate contraction (or exercise)-induced glucose uptake. Conflicting results have been reported regarding exerciseinduced fatty acid oxidation. The role of AMPK in contractioninduced fatty acid oxidation has been estimated with the use of an activator of AMPK, 5-aminoimidazole-4-carboxamide-1- - D-ribofuranoside (AICAR). Like exercise, AICAR leads to phosphorylation of acetyl-coa carboxylase 2 (ACC2), a major isoform of ACC in skeletal muscle, which decreases malonyl- CoA levels, releasing the inhibition of uptake of fatty acids into mitochondria via CPT1 and thereby stimulating fatty acid oxidation (26). In cardiac myocytes, AICAR induced translocation of fatty acid translocase (FAT)/CD36 to the sarcolemma, leading to enhanced rates of long-chain fatty acid uptake (23). However, it is not clear whether the effects of AICAR-induced increase in fatty acid oxidation are mediated solely by AMPK activation. EDL muscles were excised from AMPK mutant-overexpression Tg-Prkag3 225Q mice, AMPK 3 knockout mice, and WT mice after a swim bout and incubated for 2 h to determine the rate of oleate oxidation after exercise (3). Under these ex vivo conditions, similar rates of oleate oxidation were observed among genotypes. In this study, we sought to examine the role of AMPK activation on fatty acid oxidation during exercise by generating transgenic mice overexpressing a DN form of the 1 -AMPK subunit in skeletal muscle. We hypothesized that the exerciseinduced increase in fatty acid oxidation would be somewhat dependent on activation of AMPK. METHODS Transgenic mice. The human -skeletal actin promoter was used to drive skeletal muscle-specific expression of a DN mutant (D157A) rat 1-AMPK subunit transgene (6, 27). A complete rat 1-AMPK (GenBank Accession No. NM019142) cdna was obtained by PCR of first-strand cdna from rat skeletal muscle total RNA and subcloned into pcr2.1-topo (Invitrogen, Carlsbad, CA). Forward and reverse primer sequences were 5 -CGGAATTCATGGCCGAGAAGCA- GAAGCACGAC-3 and 5 -ATAAGAATGCGGCCGCTTACTGTG- CAAGAATTTT-3, respectively. In vitro mutagenesis (Quick Change site-directed mutagenesis kit; Stratagene, La Jolla, CA) was used to change residue Asp 157 to Ala. Asp 157 lies in the conserved DFG motif (subdomain VII in the protein kinase catalytic subunit), which is essential for Mg 2 ATP binding in all protein kinases (18, 35). It is reported that coexpression of this mutant with 1 and 1 in CCL13 cells yields a catalytically inactive complex (35) and that an 1-AMPK-DN mutant inhibits 2-catalytic activity in COS7 cells (11), rat hepatocytes (44), and mouse skeletal muscles (13). The oligonucleotides used were 5 -GAATGCAAAGATAGCCGCCT- TCGGTCTTTCAAAC-3 and 5 -GTTTGAAAGACCGAAGGCG- GCTATCTTTGCATTC-3. The 1-AMPK (D157A) cdna released from pcr2.1-topo by EcoRI and NotI digestion was subcloned into human -skeletal actin promoter plasmids. The nucleotide sequence of the 1-AMPK (D157A) cdna was confirmed by sequencing. The transgene construct contains nucleotides (nt) 2,000 to 200 of the human -skeletal actin promoter, the 1,647-bp complete rat 1- AMPK (D157A) cdna, and a polyadenylation signal that is encoded by the bovine growth hormone gene. The purified transgene fragment digested with SnaBI and SphI was microinjected into BDF1 mouse eggs at Japan SLC (Hamamatsu, Japan). Two integration-positive mouse lines, C and E, were studied. Male chimeras harboring the 1-AMPK (D157A) transgene were mated with C57BL/6J females to obtain F1 offspring. The heterozygous F1 male offspring from this breeding were then crossed with purebred C57BL/6J females to obtain heterozygous F2 offspring, and this process was continued until the heterozygous F3 generation of mice was obtained. Heterozygous 1-AMPK-DN mice and their WT littermates were compared. Mice were exposed to a cycle of 12-h light ( ) and 12-h darkness ( ) and maintained at a constant temperature of 22 C. The mice were fed a normal chow diet (CE2; CLEA Japan, Tokyo, Japan) ad libitum. All animal procedures were reviewed and approved by the National Institute of Health and Nutrition Ethics Committee on Animal Research. Western blot. The AMPK protein level in gastrocnemius was measured by Western blotting with anti- 1,- 2 (cat. no and , respectively; Upstate Biotechnology, Lake Placid, NY), - 1 (cat. no. 4182; Cell Signaling Technology, Beverly, MA), - 2 (cat. no. sc-20164; Santa Cruz Biotechnology, Santa Cruz, CA), - 1 (cat. no. 4187; Cell Signaling Technology), - 2 (cat. no. 2536; Cell Signaling Technology), and - 3 (cat. no. sc-19145; Santa Cruz Biotechnology)- AMPK antibodies. Phosphorylated and total acetyl-coa carboxylase (ACC) protein was measured by Western blotting with anti-phospho ACC (S79) antibody (cat. no ; Upstate Biotechnology) and anti-acc antibody (cat. no. 3662; Cell Signaling Technology), respectively. Measurement of AMPK activity. Isoform-specific AMPK ( 1 and 2) activity was measured as described previously (5) with antibodies against the 1-or 2-catalytic subunits of AMPK (cat. no and , respectively; Upstate Biotechnology) and Dynabeads Protein G (Dynal Biotech ASA, Oslo, Norway). Measurement of oxygen consumption and carbon dioxide production. Open-circuit indirect calorimetry was performed with an O 2/CO 2 metabolism measuring system for small animals (MK-5000RQ; Muromachi Kikai, Tokyo, Japan). The system monitored V O2 and V CO2 at 3-min intervals and calculated the respiratory quotient (RQ) ratio (V CO2/V O2). To measure energy expenditure and spontaneous motor activity when sedentary, mice were individually placed in the chamber equipped with Supermex (Muromachi Kikai) at 1630 with an adequate amount of normal chow diet. The measurements of energy expenditure under ad libitum conditions were performed from 1900 to 0700 for the dark period and from 0700 to 1630 for the light period. During fasting experiments, the remaining food was removed at 1700 and the measurements were performed while the animals were fasting from 1900 to 0700 (dark conditions) and from 0700 to 1630 (light conditions). For the exercise experiments, mice were allowed to acclimatize to the air-tight treadmill chamber (Muromachi Kikai) for 30 min, at which point V O2 and V CO2 were stable, and measurements were continued for another 30 min while mice were in a sedentary state. Mice were then exercised for 30 min at a speed of 10 m/min (low-intensity exercise). The substrate utilization rate and energy production rate were calculated using the formula used by Ferrannini (12) where the rate of glucose oxidation (g/min) 4.55 V CO2 (l/min) 3.21 V O2 (l/min) 2.87 N (mg/min), the rate of lipid oxidation (g/min) 1.67 (V O2 V CO2) 1.92 N, and the rate of energy production (kcal/min) 3.91 V O V CO N, where N is the rate of urinary nitrogen excretion used to estimate protein oxidation. However, considering

3 E49 that only a small portion of resting and exercise energy expenditure arises from protein oxidation (40), the contributions of protein oxidation were neglected. Palmitate oxidation in isolated muscle. To examine palmitate oxidation in muscles, soleus and EDL muscles were dissected tendon to tendon and placed in a 20-ml glass reaction vial containing 2 ml of warmed (30 C), pregassed (95% O 2-5% CO 2, ph 7.4), modified Krebs-Henseleit buffer containing 4% FA-free BSA (Sigma Chemical, St. Louis, MO), 5 mm glucose, and 0.5 mm palmitate, giving a palmitate-to-bsa molar ratio of 1:1. After a 30-min preincubation period, muscle strips were transferred to vials containing 0.5 Ci/ml [1-14 C]palmitate (GE Healthcare Life Sciences, Buckinghamshire, UK) for 60 min. During this phase, exogenous palmitate oxidation was monitored by the production of 14 CO 2 (7). Glycogen measurement. Muscle glycogen content was measured as glycosyl units after acid hydrolysis (22). AICAR, glucose, and insulin tolerance tests. For the AICAR tolerance test, AICAR (Toronto Research Chemicals, Toronto, Canada) was injected intraperitoneally (250 g/g body wt) into fed mice. Blood glucose levels were measured at 0, 15, 30, 60, and 90 min after AICAR injection. For the oral glucose tolerance test, D-glucose [1 mg/g body wt, 10% (wt/vol) glucose solution] was administered via a stomach tube after an overnight fast. For the insulin tolerance test, human insulin (Humulin R; Eli Lilly Japan K.K., Kobe, Japan) was injected intraperitoneally (0.75 mu/g body wt) into fed animals. Blood samples were obtained by cutting the tail tip. Blood glucose concentration was measured with a glucose analyzer (Glucometer DEx; Bayer Medical, Tokyo, Japan). Statistical analysis. Data were analyzed by one-way or two-way ANOVA. Where differences were significant, each group was compared with the other by Student s t-test (StatView 5.0; Abacus Concepts, Berkeley, CA). AICAR tolerance is plotted with respect to time and compared by two-way repeated measures ANOVA (Stat- View 5.0). In the exercise tolerance test, a Kaplan-Meier survival curve was obtained, and the comparison of groups was performed using the log-rank test. Statistical significance was defined as P Values are shown as means SE. RESULTS Production of 1 -AMPK-DN mice. 1 -AMPK-DN mice were made with a DNA construct containing the 5 -flanking skeletal muscle-specific regulatory region and promoter of the human -skeletal actin gene and a cdna encoding a DN mutant of 1 -AMPK (Fig. 1A). To examine whether overexpression of 1 -AMPK-DN impairs AMPK activity in skeletal muscle, isoform-specific AMPK ( 1 and 2 ) activity in skeletal muscle was measured (Fig. 1B). 1 -AMPK activities were 58 and 36% lower in lines C and E, respectively, than in WT Fig. 1. Generation of transgenic mice with skeletal muscle-specific overexpression of a dominant-negative (DN) form of 1-AMP-activated protein kinase (AMPK) (D157A). A: map of the DN 1-AMPK (D157A) transgene construct used for microinjection of fertilized eggs. B: activities of the 1- and 2-AMPK subunits in skeletal muscle (gastrocnemius) from lines C and E transgenic mice and wild-type (WT) littermates. AMPK activity was measured in immunoprecipitates. Values, expressed as a percentage of values in WT littermates, are means SE of 4 mice from the C line (12 wk old) and 3 mice from the E line (15 wk old). *P 0.05; ***P vs. WT littermates. C: typical data of Western blot analyses of AMPK isoforms, acetyl-coa carboxylase (ACC), and phosphorylated ACC in skeletal muscle (gastrocnemius) from 1-AMPK-DN (lines C and E) mice and WT littermates. D: 5-aminoimidazole- 4-carboxamide-1- -D-ribofuranoside (AICAR) tolerance tests. E: oral glucose tolerance tests. F: insulin tolerance tests. *P 0.05; **P 0.01; ***P vs. WT mice. Data are means SE of 3 4 mice. Male mice were used at 8 and 14 wk of age for line C and line E, respectively. For some data points, error bars are smaller than symbols.

4 E50 littermates, and 2 activities were reduced by 98 and 99%, respectively. Inhibition of 2 activity in 1 -AMPK-DN mice corresponded with that previously reported (13). Liver AMPK activity was not altered in 1 -AMPK-DN mice (data not shown). Immunoblot analysis with an isoform-specific anti- 1 - AMPK antibody showed that endogenous 1 -AMPK protein in gastrocnemius was very low in WT littermates, whereas the level of the same size 1 -AMPK protein was markedly increased in 1 -AMPK-DN mice (Fig. 1C). Although this antibody did not distinguish between the endogenous and mutated 1 -AMPK protein, these data suggested that almost all endogenous 1 -protein was replaced by mutant 1 -AMPK in 1 - AMPK-DN mice. The amount of the 63-kDa 2 -AMPK protein was decreased by 50 60% in 1 -AMPK-DN mice, which is in agreement with the results of previous studies (13, 44). The 2 -AMPK protein might be degraded, possibly due to the lack of association with - and -isoforms. The levels of other isoforms of AMPK, 1, 2, 1, and 3, were not altered in 1 -AMPK-DN mice. The 2 isoform was undetectable (data not shown). Phosphorylated ACC protein levels in 1 - AMPK-DN mice were over 60% lower than those in WT littermates (Fig. 1C), suggesting that overexpression of 1 - AMPK-DN impairs AMPK activity and subsequent phosphorylation of ACC in skeletal muscle. However, ACC protein in 1 -AMPK-DN mice (both lines of mice) was 1.5-fold larger than that in WT littermates (P 0.05; n 4 in each line of mice). The mechanism behind this is not clear. Injection of AICAR, an activator of AMPK, reduces blood glucose levels (15). In previous studies, 2 -AMPK-DN mice (13, 28) and 2 -AMPK-knockout mice (20) were resistant to the effects of AICAR. As expected, 1 -AMPK-DN mice were resistant to AICAR stimulation (Fig. 1D), indicating that AICAR-mediated activation of AMPK in skeletal muscle was severely impaired in 1 -AMPK-DN mice. However, abnormalities were not seen in the glucose and insulin tolerance curve of 1 - AMPK-DN mice (Figs. 1, E and F). Indirect calorimetry under ad libitum feeding. To examine whether substrate utilization was altered in 1 -AMPK-DN mice, 1 -AMPK-DN (line C) mice and WT littermates were subjected to measurements of oxygen consumption and RQ ratio in sedentary mice fed ad libitum (Fig. 2). The oxygen consumption (Fig. 2A, left) and RQ ratio (Fig. 2B, left) were not different between 1 -AMPK-DN mice and WT littermates during the dark cycle (feeding period) and the light cycle (sleeping period). Spontaneous motor activity was reduced during the sleeping period in both groups of mice, but it was not altered between 1 -AMPK-DN and WT littermates (Fig. 2C, left). Indirect calorimetry under fasting condition. Fasting reduces glucose oxidation in skeletal muscles and predominates fat oxidation (8). Fasting might uncover a possible abnormality in the fatty acid oxidation by 1 -AMPK-DN mice. Mice were fasted, and their oxygen consumption and RQ ratio were measured during the dark cycle and the light cycle (Fig. 2, A, B, and C, right). Oxygen consumption and RQ ratio during fasting were reduced in both groups of mice, compared with ad libitum feeding, but there was no discernable difference between both groups of mice. Body weights were reduced similarly between the two groups after 24-h fasting. These data suggested that 2 -AMPK also did not affect the fatty acid oxidation during fasting. Fig. 2. Oxygen consumption, respiratory quotient (RQ) ratio, and spontaneous motor activity while sedentary and body weight change after 24-h fasting. Oxygen consumption (A), RQ ratio (B), and spontaneous motor activity (C) were measured by open-circuit indirect calorimetry using O 2/CO 2 metabolism measuring system for small animals equipped with an infrared sensor. D: body weights were measured before and after fasting. Data are means SE of 10 male mice from 1-AMPK-DN (C line) and 8 male mice from WT (20 wk old). No significant difference is observed between 1-AMPK-DN vs. WT littermates. Exercise tolerance of 1 -AMPK-DN mice and WT littermates. First, the ability of 1 -AMPK-DN mice to tolerate an exercise bout was examined. 1 -AMPK-DN (line C) and WT littermates were subjected to two different running intensities on a treadmill. Mice were exercised at a speed of 10 m/min (low intensity) for 30 min and then a speed of 30 m/min (high intensity). Both groups of mice performed well at 10 m/min. However, at a speed of 30 m/min, some 1 -AMPK-DN mice could not continue running for 5 min and most of them dropped out before 30 min (Fig. 3A). At a speed of 10 m/min, 1 - AMPK-DN mice and WT mice were able to run for up to 6 h with seven periods of 10 min spent at rest. To elucidate the mechanism that underlies the intolerance in a high-intensity exercise, muscle glycogen was measured (Fig. 3B). The glycogen content in 1 -AMPK-DN mice before exercise was 37% lower than that in WT littermates. Although only mice that were able to tolerate exercise were examined, at 10 min after the high-intensity exercise plus 30 min of lowintensity exercise, glycogen content was reduced by 1.12 and 1.54 mol/g wet tissue in WT littermates and 1 -AMPK-DN mice, respectively, suggesting that both groups of mice had performed a substantial amount of running. After the 6-h low-intensity exercise, we measured skeletal muscle AMPK activity in 1 -AMPK-DN (line C) mice and

5 Fig. 3. Exercise tolerance and AMPK activity after low-intensity exercise. A: endurance capacity shown as a Kaplan-Meier survival curve. Ten male mice from the 1-AMPK-DN (line C) and 8 male mice from the WT littermates (each 20 wk old) were exercised by forced running on a treadmill for 30 min at 10 m/min (low-intensity) and then at 30 m/min (high-intensity) for 30 min or until exhaustion. Significant difference (P 0.014, log-rank test) was observed in the endurance capacity of these mice. B: glycogen levels in gastrocnemius. Mice were killed at indicated times after initiation of exercise; 40 min indicated 10 min after high-intensity exercise (n 3 7 in each group of mice). *P 0.05; **P C: changes in 1- and 2-AMPK subunit activities in skeletal muscle (gastrocnemius) from 1-AMPK-DN (line C) mice and WT littermates after exercise. Skeletal muscles were obtained from 1-AMPK-DN and WT littermates just after 6hoflow-intensity treadmill exercise (After exercise) or during the resting state (Resting). AMPK activity was measured in immunoprecipitates. Values, expressed as percentage of values in WT littermates, are means SE of 5 mice (15 wk old). *P 0.05 vs. resting mice. P 0.01; P vs. WT littermates. E51 their WT littermates (Fig. 3C). Compared with the resting state, 6hofexercise increased 1 -AMPK activity by 2-fold and 2 -AMPK activity by 1.4-fold in WT littermates, whereas the increases in 1 -AMPK and 2 -AMPK activities in response to exercise were not observed in 1 -AMPK-DN mice. Even after a session of exercise, both 1 - and 2 -AMPK activities were still significantly (P 0.01 and P 0.001, respectively) lower in 1 -AMPK-DN (line C) mice when compared with WT mice. Similar results were observed in line E mice (data not shown). In this experiment, 1 -AMPK activity under resting conditions in 1 -AMPK-DN mice tended to be lower but was not significantly different to WT littermates. This might be due to low sample sizes, because the other six independent experiments showed statistically significant reductions in 1 - AMPK activity in 1 -AMPK-DN mice (data not shown). These data indicated that a marked inhibition of 2 -AMPK activities persisted after the session of exercise. Indirect calorimetry during exercise. To examine which substrate was preferentially utilized during exercise in 1 - AMPK-DN mice, oxygen consumption and carbon dioxide production were monitored during low-intensity exercise and the glucose and lipid oxidation rate were calculated. We examined male and female mice at 2 mo of age (Fig. 4). In a sedentary state, the oxygen consumption and carbon dioxide production did not differ between WT littermates and 1 - AMPK-DN mice in either male or female specimens. Although data obtained at the beginning of exercise were affected by rapid gas exchange in the lung, the low-intensity exercise increased both the oxygen consumption and carbon dioxide production by 1.2- to 1.4-fold in both WT and 1 -AMPK-DN mice, irrespective of sex. Calculated RQ ratio, glucose oxidation rate, lipid oxidation rate, and energy production rate while the mice were sedentary and during exercise did not differ between the WT and 1 -AMPK-DN mice in either male or female specimens. We repeated the same experiments on mice at 6 mo of age and found that both groups of mice could increase lipid oxidation in response to low-intensity exercise (data not shown). There were no significant differences in exercise effects on plasma free fatty acids (FFA) concentrations between WT littermates and 1 -AMPK-DN mice. After 1 h of low-intensity exercise, FFA concentrations were increased from to meq/l (n 7) in WT littermates and from to (n 6) in 1 -AMPK-DN mice, respectively. These data suggest that while sedentary and during low-intensity exercise, glucose and lipid oxidation did not differ in between WT littermates and 1 -AMPK-DN mice. Palmitate oxidation in isolated skeletal muscles. To measure the rate of fatty acid oxidation in skeletal muscle during exercise directly, production of 14 CO 2 from [ 14 C]palmitate was measured ex vivo using isolated muscle obtained immediately after low-intensity exercise (10 m/min) for 30 min (Fig. 5). In soleus muscle (type I, slow-twitch muscle), the basal rate of palmitate oxidation was not impaired in 1 -AMPK-DN mice. Low-intensity exercise increased the rate of palmitate oxidation by 1.3-fold both in WT littermates and 1 -AMPK-DN mice. The increase in fatty acid oxidation was observed in another type of skeletal muscle, EDL, known as a type II rich fiber muscle. In EDL muscle, the increase in palmitate oxidation in response to low-intensity exercise was 1.4-fold. There was no discernable difference between WT littermates and

6 E52 Fig. 4. Calculated glucose and lipid oxidation rate and energy production rate during low-intensity exercise in WT littermates and 1-AMPK-DN mice. Male (A) and female (B) 1-AMPK-DN (C line) and WT (2 mo old) mice started on the treadmill at a speed of 10 m/min (low-intensity exercise) at time 0. Oxygen consumption and carbon dioxide production were monitored using an O 2/CO 2 metabolism measuring system for small animals equipped with air-tight treadmill chamber. Data of oxygen consumption and carbon dioxide production were collected for a total of 60 min, while mice were sedentary and during exercise each for 30-min interval. Calculated RQ ratio, glucose and lipid oxidation rate, and energy production rate are also shown. Values are means SE of 7 10 mice. Similar results were obtained in 1-AMPK-DN (line E) mice (data not shown). No significant difference was observed between 1- AMPK-DN vs. WT littermates. 1 -AMPK-DN mice for palmitate oxidation in response to low-intensity exercise. These data suggest that the deficiency of 2 -AMPK activity in skeletal muscle did not affect the rate of fatty acid oxidation in response to low-intensity exercise. DISCUSSION A low-intensity exercise session increased fatty acid oxidation in 2 -AMPK-deficient 1 -AMPK-DN transgenic mice in vivo and ex vivo, suggesting that activation of 2 -AMPK is not necessary for increased fatty acid oxidation in response to low-intensity exercise. Since changes in RQ ratio and oxygen utilization in the fasting state were not altered between 1 - AMPK-DN transgenic mice and WT littermates, 2 -AMPK in skeletal muscle might not play a major role in the shift to fatty acid oxidation from glucose oxidation under fasting conditions. A similar decrease in body weight in response to fasting (no

7 Fig. 5. Palmitate oxidation in isolated soleus (A) and EDL (B) muscle strips before and after exercise. Male 1-AMPK-DN (line C) mice and WT littermates (2 mo old) were assigned to the two groups. One group was kept sedentary, while the other group was subjected to the treadmill at a speed of 10 m/min for 30 min. Dissected muscles were immediately used for the measurement of palmitate oxidation. Values are means SE of 3 mice. *P 0.05 vs. sedentary. No significant difference is observed between 1-AMPK-DN vs. WT littermates. E53 energy intake) also suggested that energy expenditure was not altered in 1 -AMPK-DN transgenic mice. Our data are in good agreement with an observation that swimming increased oleate oxidation in EDL from AMPK mutant-overexpression Tg-Prkag3 225Q mice and AMPK 3 - knockout mice, similarly to that observed from WT mice (3). EDL is glycolytic (white, fast-twitch type II) muscles. Normal fatty acid oxidation in soleus (red, slow-twitch-type I) muscles in response to low-intensity exercise was also observed in 1 -AMPK-DN mice (Fig. 5B), suggesting that 2 -AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise, irrespective of fiber type. In humans, there was a discrepancy between ACC phosphorylation (a marker of AMPK activation) and fatty acid oxidation. A high-intensity exercise session increased ACC phosphorylation in the vastus lateralis muscle; however, fatty acid oxidation was not increased with increasing exercise intensity (9). In prolonged low-intensity exercise (45% of maximum oxygen consumption), ACC phosphorylation in the vastus lateralis muscle was reduced, whereas fatty acid oxidation increased (43). In perfused rat hindquarters, lowintensity muscle contraction by electrical stimulation of sciatic nerve induced an increase in fatty acid oxidation and a reduction in malonyl CoA muscle content without changes in AMPK activation and ACC activities, also suggesting that AMPK activation is not critical in the regulation of fatty acid oxidation during low-intensity muscle contraction (32). AICAR may increase fatty acid oxidation via AMPK activation (26), but low-intensity exercise increases fatty acid oxidation via AMPK-independent mechanism. Malonyl-CoA is a potent inhibitor of CPT1 (25). AMPK may enhance fatty acid oxidation in skeletal muscles, as in the liver, by inactivation of ACC via phosphorylation, thereby reducing the synthesis of malonyl-coa (42), and by activation of malonyl-coa decarboxylase, the enzyme converting malonyl-coa to acetyl-coa (29). The importance of ACC2 was supported from the finding that ACC2-knockout mice exhibited increased fat oxidation and reduced fat storage (1). However, malonyl-coa does not exclusively account for the reduction of all fat oxidation. CPT1 activity was also modified by cytosolic acetyl CoA, carnitine, and ph (21). Other factors that may affect fatty acid oxidation include the fatty acid concentration, proteins that regulate fatty acid transport, and blood flow (21). It is possible that these factors may contribute to an increase in fatty acid oxidation during low-intensity exercise. Activation of 2 -AMPK was not essential for increased fatty acid oxidation in response to low-intensity exercise, raising the possibility that 1 -AMPK may play a regulatory role. A substantial amount of 1 -AMPK remained in 1 -AMPK DN mice both in a sedentary state and after low-intensity exercise (Figs. 1B and 3C). It was proposed that residual 1 -AMPK activity in the 2 -AMPK-DN mice may largely stem from nonmuscle 1 -AMPK activity and that the partial reduction in 1 -AMPK activity in 2 -AMPK DN mice could reflect a marked reduction of 1 -AMPK activity in muscle cells (13, 28). If this is the case, 1 -AMPK is not essential for increased fatty acid oxidation in response to low-intensity exercise, either. However, using 1 -AMPK knockout mice, it was recently reported (17) that the increase in 1 -AMPK activity in soleus muscle was required for increased glucose uptake in response to ex vivo twitch contraction. Although it is unknown whether lowintensity twitch contraction ex vivo is relevant to low-intensity exercise in vivo, it is also conceivable that 1 -AMPK might be involved in an increased fatty acid oxidation in response to low-intensity exercise. Intolerance of exercise is observed in various metabolic conditions. In humans, depletion of muscle glycogen results in fatigue and impaired muscle performance and is a major determinant of endurance (16). However, in mice, it is shown that muscle glycogen is not essential for exercise, since glycogen null mice (the MGSKO mouse that disrupted the muscle isoform of glycogen synthase) do not exhibit impaired exercise tolerance compared with their WT littermates (31). In addition, a genetically modified mouse model (GSL30), which overaccumulates glycogen due to overexpression of a hyperactive form of glycogen synthase, does not possess improved exercise performance (30). Therefore, although we observed a reduction in glycogen content before and after exercise in 1 -AMPK-DN relative to WT littermates, it is not clear that compromised carbohydrate availability in 1 -AMPK-DN mice was a mechanism by which these animals lack high-intensity exercise tolerance. Mice with muscle-specific disruption of the gene encoding the GLUT4 have normal muscle glycogen levels but are impaired in their ability to exercise (38). Expression of

8 E54 GLUT4 measured by Northern blots in gastrocnemius did not differ between 1 -AMPK-DN mice (99 5%, n 7) and WT littermates (100 4%, n 7). Switching to more oxidative muscle fibers may lead to an increase in exercise endurance (39). Expressions of COX2 and COX4 mrna (mitochondrial enzymes rich in oxidative muscle fibers) in gastrocnemius did not differ between 1 -AMPK-DN mice and WT littermates in this study [COX2: 100 2% (n 7) in WT littermates and 113 2% (n 7) in 1 -AMPK-DN mice; COX4: 100 4% (n 7) in WT littermates and 110 1% (n 7) in 1 -AMPK-DN mice]. The reason for intolerance in highintensity exercise observed in 1 -AMPK-DN mice is unclear at present. In humans, peripheral lipolysis was stimulated maximally at the lowest exercise intensity (25% of maximal oxygen consumption), whereas plasma glucose tissue uptake and muscle glycogen oxidation increased in relation to exercise intensity (34). However, prolonged low-intensity exercise (30% of maximal oxygen consumption) increased FFA oxidation progressively, while glucose oxidation was reduced (2). Therefore, fatty acid oxidation in low-intensity exercise is physiologically important for reduction of fat mass and its mechanism should be clarified. In summary, we suggest that an increased 2 - AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise. GRANTS This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo); by research grants from the Japanese Ministry of Health, Labor and Welfare (Tokyo); and by a grant from the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan. C. R. Bruce is supported by a Peter Doherty postdoctoral fellowship and M. A. 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