during low-intensity exercise

Similar documents
AMPK as a metabolic switch in rat muscle, liver and adipose tissue after exercise

Role of fatty acids in the development of insulin resistance and type 2 diabetes mellitus

Metabolism of cardiac muscle. Dr. Mamoun Ahram Cardiovascular system, 2013

Regulation of glucose transport by the AMP-activated protein kinase

THE GLUCOSE-FATTY ACID-KETONE BODY CYCLE Role of ketone bodies as respiratory substrates and metabolic signals

UNIVERSITY OF BOLTON SPORT AND BIOLOGICAL SCIENCES SPORT AND EXERCISE SCIENCE PATHWAY SEMESTER TWO EXAMINATIONS 2016/2017

Medical Biochemistry and Molecular Biology department

Glucose. Glucose. Insulin Action. Introduction to Hormonal Regulation of Fuel Metabolism

BIOL212 Biochemistry of Disease. Metabolic Disorders - Obesity

INSULIN RESISTANCE: MOLECULAR MECHANISM

Phosphorylation-activity relationships of AMPK and acetyl-coa carboxylase in muscle

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

AMPK 2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle

Oxidation of Long Chain Fatty Acids

BALANCING THE SCALES USING A NOVEL CELLULAR ENERGY SENSOR

Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise

Studies in a wide variety of cultured cells have

Metabolic integration and Regulation

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation

Exercise is an important component of the treatment

Experimental Physiology

Under most conditions, glucose transport is the

Does Nitric Oxide Regulate Skeletal Muscle Glucose Uptake during Exercise?

Manipulation of the Nutrient Sensors (AMPK/TOR) with Anaplerotic Diet Therapy (Triheptanoin) An Alternative to Diet Restriction

number Done by Corrected by Doctor Faisal Al-Khatibe

Roles of Lipids. principal form of stored energy major constituents of cell membranes vitamins messengers intra and extracellular

The AMP-activated protein kinase activator AICAR does not induce GLUT4 translocation to transverse tubules. protein kinases and in skeletal muscle

Key words: Branched-chain c~-keto acid dehydrogenase complex, branched-chain c~-keto acid

Skeletal Muscle Metabolic Flexibility: The Roles of AMP-Activated Protein Kinase and Calcineurin

Exercise and insulin stimulate glucose transport

Critical Review. Skeletal Muscle Glucose Uptake During Exercise: A Focus on Reactive Oxygen Species and Nitric Oxide Signaling

Cellular Respiration

The 5 AMP-activated protein kinase (AMPK) is a

In glycolysis, glucose is converted to pyruvate. If the pyruvate is reduced to lactate, the pathway does not require O 2 and is called anaerobic

Determine Of the Exercise Intensity That Elicits Maximal Fat Oxidation In Untrained Male Students

William G. Aschenbach, Michael F. Hirshman, Nobuharu Fujii, Kei Sakamoto, Kirsten F. Howlett, and Laurie J. Goodyear

Integration Of Metabolism

control kda ATGL ATGLi HSL 82 GAPDH * ** *** WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi iwat gwat ibat

Position: Associate Professor, Department of Molecular and Integrative Physiology

Chronic activation of 5 -AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle

IL-6 has recently been recognized as a myokine (1), which is released

Modifications of Pyruvate Handling in Health and Disease Prof. Mary Sugden

Final Review Sessions. 3/16 (FRI) 126 Wellman (4-6 6 pm) 3/19 (MON) 1309 Surge 3 (4-6 6 pm) Office Hours

Enhanced Muscle Insulin Sensitivity After Contraction/Exercise Is Mediated by AMPK

Skeletal Muscle Lipid Metabolism in Exercise and Insulin Resistance

Energy Production In A Cell (Chapter 25 Metabolism)

Test next Thursday, the 24 th will only cover the lecture

AMPK a 1 Activation Is Required for Stimulation of Glucose Uptake by Twitch Contraction, but Not by H 2 O 2, in Mouse Skeletal Muscle

The Journal of Physiology

Supplemental Table 1 Primer sequences (mouse) used for real-time qrt-pcr studies

Intermediary metabolism. Eva Samcová

AMPK activity and isoform protein expression are similar in muscle of obese subjects with and without type 2 diabetes

Supplementary Figure 1

Energy metabolism - the overview

ROLE OF AMPK SUBUNITS IN SKELETAL MUSCLE mtor SIGNALING

Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state

Biosynthesis of Fatty Acids. By Dr.QUTAIBA A. QASIM

g) Cellular Respiration Higher Human Biology

5.0 HORMONAL CONTROL OF CARBOHYDRATE METABOLISM

Supplementary Figure 1. DJ-1 modulates ROS concentration in mouse skeletal muscle.

Supplementary Table 1. Primer Sequences Used for Quantitative Real-Time PCR

Sustained hyperleptinemia induced in normal rats by adenovirus

BCM 221 LECTURES OJEMEKELE O.

Interaction of diet and training on endurance performance in rats

AMPK. Tomáš Kučera.

Skeletal muscle AMPK is essential for the maintenance of FNDC5 expression

NEW METHODS FOR ASSESSING SUBSTRATE UTILIZATION IN HORSES DURING EXERCISE

5-Aminoimidazole-4-carboxamide-1-

AMPK Phosphorylation Assay Kit

number Done by Corrected by Doctor Nayef Karadsheh

9/17/2009. HPER 3970 Dr. Ayers. (courtesy of Dr. Cheatham)

Fatty acid breakdown

UNIVERSITY OF PNG SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

Allometry. The Problem of Size & Scaling. Get it??? A LLAMA TREE

Integrative Metabolism: Significance

Insulin Signaling After Exercise in Insulin Receptor Substrate-2 Deficient Mice

AMPK. Tomáš Kuc era. Ústav lékar ské chemie a klinické biochemie 2. lékar ská fakulta, Univerzita Karlova v Praze

LIMITS TO HUMAN ENDURANCE: CARNITINE AND FAT OXIDATION. Francis B. Stephens, University of Nottingham. Stuart D.R. Galloway, University of Stirling

Overall Energy metabolism: Integration and Regulation

Title. CitationLife Sciences, 88(3-4): Issue Date Doc URL. Type. File Information.

EXERCISE PRESCRIPTION FOR OBESE PATIENT

1st half of glycolysis (5 reactions) Glucose priming get glucose ready to split phosphorylate glucose rearrangement split destabilized glucose

Supplementary Figure 1

Implications of mitochondrial skeletal muscle metabolism on diabetes and obesity before and after weight loss

Fuel the Failing Heart: glucose or fatty acids? Rong Tian, MD, PhD Mitochondria and Metabolism Center University of Washington, Seattle

UNIVERSITY OF BOLTON SCHOOL OF SPORT AND BIOMEDICAL SCIENCES SPORT PATHWAYS WITH FOUNDATION YEAR SEMESTER TWO EXAMINATIONS 2015/2016

Moh Tarek. Razi Kittaneh. Jaqen H ghar

LIPID METABOLISM

Presented by: Mariam Boulas Veronica Dascalu Pardis Payami

PHY MUSCLE AND EXERCISE. LECTURE 2: Introduction to Exercise Metabolism

Muscle Metabolism. Dr. Nabil Bashir

J.D. Pagan*, B. Essen-Gustavsson, A. Lindholm, and J. Thornton

Integration of Metabolism 1. made by: Noor M. ALnairat. Sheet No. 18

Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise

2/25/2015. Anaerobic Pathways. Glycolysis. Alternate Endpoints. Gluconeogenesis fate of end products

Role of the Pyruvate

Tala Saleh. Razi Kittaneh ... Nayef Karadsheh

Effect of different types of carbohydrate supplementation on glycogen supercompensation in rat skeletal muscle

Food a fact of life eseminar: ENERGY REQUIREMENTS FOR SPORT. Dr Sarah Schenker British Nutrition Foundation

AMPK- 2 is involved in exercise training-induced adaptations in insulinstimulated metabolism in skeletal muscle following high-fat diet

Transcription:

Am J Physiol Endocrinol Metab 296: E47 E55, 2009. First published October 21, 2008; doi:10.1152/ajpendo.90690.2008. 2 -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, 2009. First published October 21, 2008; doi:10.1152/ajpendo.90690.2008. 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, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8636, Japan (e-mail: 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. http://www.ajpendo.org 0193-1849/09 $8.00 Copyright 2009 the American Physiological Society E47

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 (0700-1900) and 12-h darkness (1900-0700) 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. 07-350 and 07-363, 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. 07-303; 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. 07-350 and 07-363, 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 O2 1.10 V CO2 3.34 N, where N is the rate of urinary nitrogen excretion used to estimate protein oxidation. However, considering

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 0.05. 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 0.001 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 0.001 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.

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

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 0.01. 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 0.001 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 0.37 0.06 to 0.53 0.04 meq/l (n 7) in WT littermates and from 0.28 0.04 to 0.48 0.08 (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

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

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

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. Febbraio a Principal Research Fellowship from the National Health and Medical Research Council of Australia. REFERENCES 1. Abu-Elheiga L, Matzuk MM, Abo-Hashema KA, Wakil SJ. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl- CoA carboxylase 2. Science 291: 2613 2616, 2001. 2. Ahlborg G, Felig P, Hagenfeldt L, Hendler R, Wahren J. Substrate turnover during prolonged exercise in man. Splanchnic and leg metabolism of glucose, free fatty acids, and amino acids. J Clin Invest 53: 1080 1090, 1974. 3. Barnes BR, Long YC, Steiler TL, Leng Y, Galuska D, Wojtaszewski JF, Andersson L, Zierath JR. Changes in exercise-induced gene expression in 5 -AMP-activated protein kinase gamma3-null and gamma3 R225Q transgenic mice. Diabetes 54: 3484 3489, 2005. 4. Birk JB, Wojtaszewski JF. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol 577: 1021 1032, 2006. 5. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mtor) signaling. J Biol Chem 277: 23977 23980, 2002. 6. Brennan KJ, Hardeman EC. Quantitative analysis of the human alphaskeletal actin gene in transgenic mice. J Biol Chem 268: 719 725, 1993. 7. Bruce CR, Brolin C, Turner N, Cleasby ME, van der Leij FR, Cooney GJ, Kraegen EW. Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification. Am J Physiol Endocrinol Metab 292: E1231 E1237, 2007. 8. Cahill GF Jr. Starvation in man. N Engl J Med 282: 668 675, 1970. 9. Chen ZP, Stephens TJ, Murthy S, Canny BJ, Hargreaves M, Witters LA, Kemp BE, McConell GK. Effect of exercise intensity on skeletal muscle AMPK signaling in humans. Diabetes 52: 2205 2212, 2003. 10. Douen AG, Ramlal T, Rastogi S, Bilan PJ, Cartee GD, Vranic M, Holloszy JO, Klip A. Exercise induces recruitment of the insulinresponsive glucose transporter. Evidence for distinct intracellular insulinand exercise-recruitable transporter pools in skeletal muscle. J Biol Chem 265: 13427 13430, 1990. 11. Dyck JR, Gao G, Widmer J, Stapleton D, Fernandez CS, Kemp BE, Witters LA. Regulation of 5 -AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J Biol Chem 271: 17798 17803, 1996. 12. Ferrannini E. The theoretical bases of indirect calorimetry: a review. Metabolism 37: 287 301, 1988. 13. Fujii N, Hirshman MF, Kane EM, Ho RC, Peter LE, Seifert MM, Goodyear LJ. AMP-activated protein kinase 2 activity is not essential for contraction-and hyperosmolarity-induced glucose transport in skeletal muscle. J Biol Chem 280: 39033 39041, 2005. 14. Goodyear LJ, Hirshman MF, Horton ES. Exercise-induced translocation of skeletal muscle glucose transporters. Am J Physiol Endocrinol Metab 261: E795 E799, 1991. 15. Hardie DG, CarlingD, Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67: 821 855, 1998. 16. Holloszy JO, Kohrt WM, Hansen PA. The regulation of carbohydrate and fat metabolism during and after exercise. Front Biosci 3: D1011 D1027, 1998. 17. Jensen TE, Schjerling P, Viollet B, Wojtaszewski JF, Richter EA. AMPK alpha1 activation is required for stimulation of glucose uptake by twitch contraction, but not by H2O2, in mouse skeletal muscle. PLoS ONE 3: e2102, 2008. 18. Johnson LN, Noble ME, Owen DJ. Active and inactive protein kinases: structural basis for regulation. Cell 85: 149 158, 1996. 19. Jorgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA, Wojtaszewski JF. Knockout of the alpha2 but not alpha1 5 -AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279: 1070 1079, 2004. 20. Jorgensen SB, Wojtaszewski JF, Viollet B, Andreelli F, Birk JB, Hellsten Y, Schjerling P, Vaulont S, Neufer PD, Richter EA, Pilegaard H. Effects of alpha-ampk knockout on exercise-induced gene activation in mouse skeletal muscle. FASEB J 19: 1146 1148, 2005. 21. Kiens B. Skeletal muscle lipid metabolism in exercise and insulin resistance. Physiol Rev 86: 205 243, 2006. 22. Lowry OH, Passonneau JV. A Flexible System of Enzymatic Analysis. London: Academic, 1972, p. 1 291. 23. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, Glatz JF. Contraction-induced fatty acid translocase/cd36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627 1634, 2003. 24. Mahlapuu M, Johansson C, Lindgren K, Hjälm G, Barnes BR, Krook A, Zierath JR, Andersson L, Marklund S. Expression profiling of the -subunit isoforms of AMP-activated protein kinase suggests a major role for 3 in white skeletal muscle. Am J Physiol Endocrinol Metab 286: E194 E200, 2004. 25. McGarry JD, Mills SE, Long CS, Foster DW. Observations on the affinity for carnitine, and malonyl-coa sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-coa in non-hepatic tissues of the rat. Biochem J 214: 21 28, 1983. 26. Merrill GF, Kurth EJ, Hardie DG, Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol Endocrinol Metab 273: E1107 E1112, 1997. 27. Miura S, Kai Y, Ono M, Ezaki O. Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mrna in skeletal muscles. J Biol Chem 278: 31385 31390, 2003. 28. Mu J, Brozinick JT Jr, Valladares O, Bucan M, Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7: 1085 1094, 2001. 29. Park H, Kaushik VK, Constant S, Prentki M, Przybytkowski E, Ruderman NB, Saha AK. Coordinate regulation of malonyl-coa decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-coa carboxylase by AMP-activated protein kinase in rat tissues in response to exercise. J Biol Chem 277: 32571 32577, 2002. 30. Pederson BA, Cope CR, Irimia JM, Schroeder JM, Thurberg BL, Depaoli-Roach AA, Roach PJ. Mice with elevated muscle glycogen

E55 stores do not have improved exercise performance. Biochem Biophys Res Commun 331: 491 496, 2005. 31. Pederson BA, Cope CR, Schroeder JM, Smith MW, Irimia JM, Thurberg BL, DePaoli-Roach AA, Roach PJ. Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise. J Biol Chem 280: 17260 17265, 2005. 32. Raney MA, Yee AJ, Todd MK, Turcotte LP. AMPK activation is not critical in the regulation of muscle FA uptake and oxidation during low-intensity muscle contraction. Am J Physiol Endocrinol Metab 288: E592 E598, 2005. 33. Rasmussen BB, Wolfe RR. Regulation of fatty acid oxidation in skeletal muscle. Annu Rev Nutr 19: 463 484, 1999. 34. Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, Wolfe RR. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol Endocrinol Metab 265: E380 E391, 1993. 35. Stein SC, Woods A, Jones NA, Davison MD, Carling D. The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345: 437 443, 2000. 36. Toyoda T, Tanaka S, Ebihara K, Masuzaki H, Hosoda K, Sato K, Fushiki T, Nakao K, Hayashi T. Low-intensity contraction activates the alpha1-isoform of 5 -AMP-activated protein kinase in rat skeletal muscle. Am J Physiol Endocrinol Metab 290: E583 E590, 2006. 37. Viollet B, Andreelli F, Jorgensen SB, Perrin C, Geloen A, Flamez D, Mu J, Lenzner C, Baud O, Bennoun M, Gomas E, Nicolas G, Wojtaszewski JF, Kahn A, Carling D, Schuit FC, Birnbaum MJ, Richter EA, Burcelin R, Vaulont S. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity. J Clin Invest 111: 91 98, 2003. 38. Wallberg-Henriksson H, Zierath JR. GLUT4: A key player regulating glucose homeostasis? Insights from transgenic and knockout mice (review). Mol Membr Biol 18: 205 211, 2001. 39. Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang H, Evans RM. Regulation of muscle fiber type and running endurance by PPARdelta. PLoS Biol 2: e294, 2004. 40. Weir JB. New methods for calculating metabolic rate with special reference to protein metabolism. J Physiol 109: 1 9, 1949. 41. Winder WW. Intramuscular mechanisms regulating fatty acid oxidation during exercise. Adv Exp Med Biol 441: 239 248, 1998. 42. Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, Call GB, Clayton RD, Conley LM, Yoon S, Zhou B. Phosphorylation of rat muscle acetyl-coa carboxylase by AMP-activated protein kinase and protein kinase A. J Appl Physiol 82: 219 225, 1997. 43. Wojtaszewski JF, Mourtzakis M, Hillig T, Saltin B, Pilegaard H. Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise. Biochem Biophys Res Commun 298: 309 316, 2002. 44. Woods A, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P, Foufelle F, Carling D. Characterization of the role of AMPactivated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol Cell Biol 20: 6704 6711, 2000. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on November 26, 2017