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1 J Physiol (215) pp AMP activated protein kinase α 2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4 Andreas Mæchel Fritzen 1, Anne-Marie Lundsgaard 1,4,JacobJeppesen 1,3, Mette Landau Brabæk Christiansen 1, Rasmus Biensø 2,JasonR.B.Dyck 5, Henriette Pilegaard 2 and Bente Kiens 1 The Journal of Physiology 1 Section of Molecular Physiology, Department of Nutrition, and Sports, the August Krogh Centre, University of Copenhagen, Copenhagen, Denmark 2 Centre of Inflammation and Metabolism, the August Krogh Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark 3 Type 2 Diabetes and Obesity Pharmacology, Novo Nordisk A/S, Maaloev, Denmark 4 Danish Diabetes Academy, Odense University Hospital, Odense, Denmark 5 Cardiovascular Research Centre, Alberta Diabetes Institute, Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada Key points There is lower fat oxidation during post-exercise recovery in mice lacking 5 -AMP activated protein kinase α 2 (AMPKα 2 ). AMPKα2 is involved in post-transcriptional and not transcriptional regulation of pyruvate dehydrogenase kinase 4 (PDK4) in muscle. -induced AMPKα2 activity increases PDK4 protein content, in turn inhibiting pyruvate dehydrogenase activity and glucose oxidation. The mechanism for increased post-exercise fat oxidation is by inhibition of carbohydrate oxidation allowing increased fat oxidation rather than by direct stimulation of fat oxidation. Abstract It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post-exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post-exercise in 5 -AMP activated protein kinase (AMPK) α 2 and α 1 knock-out (KO) and wild-type () mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post-exercise recovery, a lower lipid oxidation (P <.5) and higher glucose oxidation were observed in AMPKα 2 KO (respiratory exchange ratio (RER) =.84 ±.2) than in and AMPKα 1 KO (average RER =.8 ±.1) without genotype differences in muscle malonyl-coa or free-carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mrna expression in and AMPKα 2 KO was observed following exercise, which is consistent with AMPKα 2 deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P <.1) in but remained unchanged in AMPKα 2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P <.1) inhibitory pyruvate dehydrogenase (PDH)-E1α Ser 293 phosphorylation was observed in AMPKα 2 KO muscle compared to. These findings indicate that AMPKα 2 regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation. DOI: /JP27821

2 4766 A. M. Fritzen and others J Physiol (Resubmitted 15 June 215; accepted after revision 2 September 215; first published online 11 September 215) Corresponding author B. Kiens: Department of Nutrition, and Sports, the August Krogh Centre, University of Copenhagen, Universitetsparken 13, 21 Copenhagen Ø, Denmark. bkiens@nexs.ku.dk Abbreviations ACC, acetyl-coa carboxylase; AICAR, 5-aminoimidazole-4-carboxamide riboside; AMPK, 5 -AMP activated protein kinase; C T, cycle threshold; FA, fatty acid; HDAC4, histone deacetylase 4; KO, knock-out; LPL, lipoprotein lipase; mir, micro ribonucleic acid; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase isoenzyme 4; RER, respiratory exchange ratio; SIRT1, silent information regulator T1; ssdna, single stranded DNA; TG, triacylglycerol;, wild-type. Introduction Several human studies have shown a substantial increase in fatty acid (FA) oxidation in the post-exercise period compared to the resting state. Depending on the intensity and duration of exercise, the enhanced FA oxidation will last for several hours or even days after exercise (Krzentowski et al. 1982; Bielinski et al. 1985; Maehlum et al. 1986; Weststrate et al. 199; Wolfe et al. 199; Melby et al. 1993; Calles-Escandon et al. 1996; Treuth et al. 1996; Horton et al. 1998; Kiens & Richter, 1998; Kimber et al. 23). Even with an increaseddietary intake of carbohydrates post-exercise, where a high carbohydrate oxidation would be expected, the contribution of FA for oxidation is high (Bielinski et al. 1985; Kiens & Richter, 1998; Kimber et al. 23). It appears that muscle glycogen resynthesis after exercise has such a high metabolic priority that utilization of lipids is elevated to cover the energy expenditure in muscle cells (Kiens & Richter, 1998; Kimber et al. 23). However, the mechanisms regulating the selection of energy substrate towards oxidation or storage in muscle during recovery from exercise remain unsolved. The heterotrimeric AMP-activated protein kinase complex (AMPK), consisting of a catalytic α-subunit (α 1 or α 2 ) in combination with a regulatory β- (β 1 or β 2 )andγ-subunit (γ 1, γ 2 or γ 3 ), is activated by increased AMP:ATP and ADP:ATP ratios and stimulates substrate metabolism (Hardie, 28; Richter & Ruderman, 29). A role of AMPK in the regulation of FA oxidation during post-exercise recovery has been suggested from studies in rats (Rasmussen et al. 1998). Here it was shown that malonyl coenzyme A (CoA) content and acetyl-coa carboxylase (ACC) activity remained suppressed, and FA oxidation enhanced, in skeletal muscle for prolonged periods after submaximal exercise, suggesting a mechanism via malonyl-coa, ACC and carnitine palmitoyltransferase 1 (CPT1) that allows for the enhanced FA oxidation post-exercise (Rasmussen et al. 1998). The ability of cells to switch between glucose and FAs for oxidation can also be determined by the PDH complex. PDH catalyses the irreversible oxidative decarboxylation of pyruvate into acetyl-coa. Recent findings suggest that AMPK is engaged in the regulation of the PDH complex. Thus, Klein et al. (27) showed an increased PDH activity in skeletal muscle from AMPKα 2 knock-out (KO) mice compared with wild-type () at rest and during exercise (Klein et al. 27). Akey enzymein controllingtheactivity of PDH is pyruvate dehydrogenase kinase 4 (PDK4), which acts by inhibition of PDH activity and thereby preventing the entry of pyruvate into the Krebs cycle (Sugden et al. 1993). Furthermore, an increased transcription of PDK4 was found in primary cardiomyocytes from rats when stimulated with 5-aminoimidazole-4-carboxamide riboside (AICAR) (Houten et al. 29) pointing towards an engagement of AMPK in the regulation of PDK4. Proposed stimulators of PDK4 transcription are both silent information regulator T1 (SIRT1) and histone deacetylase 4 (HDAC4). From studies in C2C12 cells (a mouse myoblast cell line) it was suggested that AMPK stimulated SIRT1 activity (Cantó et al. 29; Cantó & Auwerx, 211), and studies in human muscle samples suggested an association between AMPK activity and HDAC4 export from the nucleus (McGee et al. 29). Together these various findings may indicate that AMPK regulates PDH activity via regulation of the upstream PDK4 in skeletal muscle at rest and during exercise. It could be speculated that the ability to switch towards an increased FA oxidation during post-exercise recovery also could be attributable to AMPK. Support for such a notion are the findings of a deactivation of PDH in human skeletal muscle in the hours following prolonged exercise (Kimber et al. 23). Accordingly, an increased FA oxidation would allow for a greater proportion of the glucose taken up being directed towards the glycogen synthesis pathway. This is in accordance with the findings that the muscle glycogen levels during post-exercise recovery, after 3 9 min of treadmill running or 2 h of swimming, were lower in mice lacking functional AMPK than in (Mu et al. 23; Barnes et al. 24; Jørgensen et al. 25). We therefore hypothesized that AMPK orchestrates the switch in fuel selection towards higher FA oxidation post-exercise by increasing PDK4 protein thereby inhibiting PDH activity. Consequently, the glucose taken up will be directed towards glycogen synthesis rather than oxidation. To test the hypothesis, and since both α 1 and α 2 AMPK-containing complexes are present in skeletal muscle and potentially could regulate fuel selection post-exercise, we investigated both AMPKα 1 and AMPKα 2 KO mice and their respective littermates in metabolic

3 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4767 chambers during, and in the period after, an acute exercise bout at the same relative intensity. Methods Ethical approval All experiments as well as the breeding protocol were approved by the Danish Animal Experimental Inspectorate and complied with the European Convention for the Protection of Vertebrate Animals used for Experiments and other Scientific Purposes. Animals To elucidate whether a role of AMPK in substrate selection post-exercise could be allocated to either AMPKα 1 or AMPKα 2 containing heterotrimers, female AMPKα 1 and AMPKα 2 whole-bodykoandtheirrespective littermates were used. Mice were weeks during the characterization experiments and weeks at their final termination. The mice were generated as previously described (Viollet et al. 23; Jørgensen et al. 24). Briefly, AMPKα 1 KO mice were generated on 129sv background, and AMPKα 2 KO mice were generated on C57Bl/6 background. In both models, homozygote and KO littermates were generated by heterozygote inter-cross breeding. AMPKα 2 KO mice have a normal lifespan with body weight, body composition and food intake similar to (Viollet et al. 23). Genotyping was performed by PCR analysis as previously described (Jørgensen et al. 24; Thomson et al. 27) and was later verified by immunoblotting. Mice were housed in temperature controlled (22 ± 1 C) facilities, maintained on a 12 h:12 h light dark cycle, and received standard chow (Altromin, cat. no. 1324; Brogaarden, Lynge, Denmark) and water ad libitum. Treadmill running exercise Mice were studied in the post-exercise recovery period after having performed a prolonged treadmill exercise test. Prior to the treadmill exercise test, all mice were acclimatized on a treadmill apparatus (TSE Systems GmbH, Germany). Acclimatization was performed two times a day, separated by 5 h of rest for 3 days. Thereafter, mice rested 2 days prior to the experimental day. At acclimatization mice first rested for 3 min on the treadmill which was followed by running at a % incline: Day 1, 5 min at increasing speed from 7 m min 1 and 5 min at 14 m min 1 ; Day 2, 5 min at 14 m min 1 and 5 min at 17 m min 1 ; Day 3, 1 min at 17 m min 1.Thereafterall mice performed a maximal running speed test, in which the mice started at 1.8 m min 1 at a % incline. Then the speed was increased by 2.4 m min 1 every 2nd minute until the mice were unable to keep up with treadmill speed. Cut-off speed was defined as the maximal running speed. Measurement of oxygen uptake and respiratory exchange ratio Oxygen uptake and CO 2 production were measured using a CaloSys apparatus (TSE Systems, Bad Homburg, Germany) at rest (24 h), during the running test and 6 h into post-exercise recovery in eight mice from each group. The respiratory exchange ratio (RER) was calculated as CO 2 production/o 2 uptake. FAT utilization was calculated as 19 kj/l O 2 oxygen uptake (l O 2 /h/kg) (1% - ((RER-.7)/.3)). For recording during rest, mice were acclimatized to individual cages for 24 h prior to the measurement. The mice were allowed access to food and water ad libitum at all times while housed in individual cages. For recording during exercise, AMPKα 1 KO and littermates as well as AMPKα 2 KO and littermates underwent 2 h of treadmill exercise at 5% of average maximal running speed per group. AMPKα 1 KO and mice ran at 16 m min 1, and AMPKα 2 KO and mice ran at 13 and 18 m min 1, respectively. After exercise the mice were transferred to individual calorimetric cages and allowed access to chow food and water ad libitum, ando 2 uptake and CO 2 production were measured during the following 6 h post-exercise recovery period. Thereafter, and AMPKα 2 KO mice were allocated into a basal, resting group (Rest 1:, n = 1; KO, n = 1) and an exercise group (, n = 1; KO, n = 1). Another group of and AMPKα 2 KO mice were allocated into a basal, resting group (:, n = 9; KO, n = 8) and a recovery group (, n = 1; KO, n = 7). In some of the biochemical analyses, only 7 8 samples were used due to either a lack of samples or technical issues. The exercise group performed 2 h of treadmill exercise at 5% of individual group maximal running speed between 16. and 18. h. The recovery group performed initially 2 h of treadmill exercise at 5% of individual group maximal running speed between 1. and 12. h and were afterwards placed in individual cages and rested for 6 h with food and water ad libitum. The resting groups (Rest 1 and ) had free access to water and food until termination. All mice were killed by cervical dislocation at 18. h, which was the same standardized time of the day for all animals. Gastrocnemius and quadriceps muscles were quickly removed and immediately frozen in liquid nitrogen and stored at 8 C until being further processed. In addition, a blood sample was collected from the chest cavity after heart puncture and transferred to Eppendorf tubes containing 3 μl of 2 mm EGTA. The blood was centrifuged in a Dich centrifuge at 18, g (type 155SLRC-BK; Ole Dich Instrumentmakers, Denmark) for 3 min, after which

4 4768 A. M. Fritzen and others J Physiol plasma was collected and stored at 2 C for further analyses. In a previous study muscle glycogen was only partially resynthesized 3 h post-exercise in muscles from mice overexpressing a kinase-dead AMPKα 2 as well as in mice (Mu et al. 23), which is why we chose to kill the mice 6 h post-exercise. Muscle and blood metabolites All reagents were from Sigma-Aldrich Denmark (Copenhagen, Denmark) unless stated otherwise. The following was measured in the quadriceps muscle: Glycogen concentration was determined on 5 mg (wet weight) pulverized tissue after acidic hydrolysis and measured spectrophotometrically at 34 nm (Hitachi 912 Automatic Analyser; Boehringer, Mannheim, Germany) (Passonneau et al. 1967; Lowry & Passonneau, 1972). Malonyl-CoA, acetyl-coa, succinyl-coa and free CoA were assayed on 1 15 mg (wet wt) pulverized muscle tissue by high-pressure liquid chromatography separation followed by ultraviolet detection as previously described (Stanley et al. 1996). Free carnitine content was determined in 1 mg of freeze-dried and dissected muscle tissue by a radioisotopic assay (Cederblad et al. 199) as described previously (Roepstorff et al. 25). NAD + and NADH content were determined on 1 mg of freeze-dried and dissected muscle tissue using a photometrical NAD/NADH quantification kit (Biovision, Mountain View, CA, USA). Plasma glucose (Wako Chemicals BmbH, Neuss, Germany), plasma fatty acids (FAs) (Wako Chemicals) and plasma triacylglycerol (TG) concentrations (triacylglycerol GPO-PAP kit, Roche Diagnostics, Mannheim, Germany) were measured using enzymatic colorimetric methods (Hitachi 912 automatic analyser; Boehringer, Mannheim, Germany). RNA isolation, reverse transcription and real time PCR Total RNA was isolated from 2 mg pulverized wet muscle tissue from the quadriceps muscle by a guanidinium thiocyanate phenol chloroform extraction method (Chomczynski & Sacchi, 1987) with modifications (Pilegaard et al. 2). Superscript II RNase H -system and oligo(dt) (Invitrogen, Carlsbad, CA, USA) were used to reverse transcribe the mrna to cdna as previously described (Pilegaard et al. 2). The cdna samples were diluted to a concentration of 6 μl μg 1 mrna in nuclease-free H 2 O. The amount of single stranded DNA (ssdna) was determined in each cdna sample using OliGreen reagent (Molecular Probes, Leiden, The Netherlands) according to Lundby et al. (26). Before reverse transcription of microrna (mir), RNA samples were diluted to 2 ng RNA μl 1. MicroRNAs were then reverse transcribed to cdna by using TaqMan Micro- RNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and mir-specific primer (Applied Biosystems). The reaction was run in a thermal cycler (PTC-2; MJ Research, Waltham, MA, USA). Real time PCR was performed with an ABI 79 sequence-detection system (Applied Biosystems) and has previously been published (Kiilerich et al. 21). Primers and TaqMan probes for amplifying a PDK4-specific mrna fragment were designed using the mouse-specific database from Ensembl ( html) and Primer Express (Applied Biosystems). The probe was 5-6-carboxyfluorescein (FAM) and 3-6- carboxy-n,n,n,n -tetramethylrhodamine (TAMRA) labelled, and primers and probes were obtained from TAG Copenhagen (Copenhagen, Denmark). The obtained cycle threshold (C T ) values reflecting the initial content of the transcript in the samples were converted to an arbitrary amount by using standard curves obtained from a serial dilution of a representative pooled sample. For each sample, the amount of a given target cdna was normalized to the ssdna content of the RT sample. The content of mir-17, RNU6B, snorna135, snorna22 and snorna234 was determined by real time PCR (as described above) using predeveloped mir assays containing specific primers and TaqMan probes labelled with 5-6-carboxyfluorescein and minor groove binder quencher (non-fluorescent) (Applied Biosystems). The obtained C T values reflecting the initial content of the measured mirs in the samples were converted to an arbitrary amount by using standard curves obtained from a serial dilution of a pooled sample made from all samples. Lipoprotein lipase activity Activity of lipoprotein lipase (LPL) was determined in vitro as previously described (Lithell & Boberg, 1978; Kiens et al. 1989). In brief, muscle samples from gastrocnemius muscle (5 mg wet wt) were incubated in a [ 3 H]triolein emulsion. During incubation LPL was released from its vascular binding to heparin sulphate proteoglycans by heparin added to the incubation medium. Moreover, human serum was added as an apolipoprotein C-II donor and albumin as an acceptor of liberated fatty acids. The release of [ 3 H]oleic acid during the last 6 min of the 11 min incubation was used as a measure of the activity of LPL. During this time, the rate of fatty acid release was linear. One nanomole of fatty acid released per minute corresponds to 1 mu LPL enzyme activity. Western blotting Total protein content, phosphorylation and acetylation levels of proteins were determined in muscle lysates. Muscle samples from the quadriceps muscle were

5 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4769 freeze-dried and dissected free of all visible adipose tissue, connective tissue and blood under a microscope, and 5 mg dry wt was homogenized in ice-cold buffer as previously described (Jeppesen et al. 21) modified with deacetylase inhibition by adding 1 mm nicotinamide and 1 mm sodium butyrate. Total protein concentration of muscle lysates was determined in triplicates using the bicinchoninic acid (BCA) method with a Pierce BCA protein assay (no ; Pierce Biotechnology, Rockford, IL, USA). A maximal coefficient of variance of 5% was accepted between replicates. All samples were heated (96 C in 5 min) in Laemmli buffer before being subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and semi-dry immunoblotting. The following antibodies against proteins were used: anti-acetyl-coa carboxylase (ACC) (streptavidin HRP, product no. P397: Dako, Glostrup, Denmark), anti-ampkα 1 and anti-ampkα 2 (kindly donated by Dr Hardie, Dundee University, UK). The following primary phospho-specific antibodies were used: anti-acc Ser 79 phosphorylation (no. 7-33, Upstate Biotechnology Incorporated, Waltham, MA, USA) (the ACC phospho-specific antibody is raised against a peptide corresponding to the sequence in rat ACC1 containing the Ser 79 phosphorylation site, but the antibody also recognises the mouse ACC2 when phosphorylated at the corresponding Ser 212 ), and anti-α-ampk Thr 172 phosphorylation (no. 2535, Cell Signaling Technology, Danvers, MA, USA). Furthermore anti-p53 (no. 2524) and anti-acetylated p53 Lys 379 (no. 257) (Cell Signaling Technology) were used. In addition, protein levels of the PDH-E1α subunit, PDH-E1α phosphorylation at Ser 293 and Ser 3 (Pilegaard et al. 26) and pyruvate dehydrogenase kinase isozyme 4 (PDK4) protein levels (Kiilerich et al. 21) were determined using antibodies as previously described. Membranes were probed with enhanced chemiluminescence (ECL + ; Amersham Biosciences, Piscataway, NJ, USA), and immune complexes were visualized using a BioRad ChemiDoc MP Imaging System (Hercules, CA, USA). Signals were quantified (Image Lab version 4., BioRad) and expressed as arbitrary units. Membranes used for detection of phosphorylated ACC and acetylated p53 were stripped with a buffer containing 1 mm 2-mercaptoethanol, 2% SDS, and 62.5 mm Tris HCl and reprobed with the corresponding antibody against the corresponding protein. Loading consistency was verified by Coomassie stain. Coomassie stain after development was performed by submersion of PVDF membranes for 1 min in.5% Coomassie Blue G-25 in 5% ethanol 1% acetic acid, removal of excess Coomassie stain with distilled water followed by destaining in 5% ethanol 1% acetic acid until bands were clearly visible. Statistics Data are expressed as means ± SEM. All statistical analyses were performed in SigmaPlot 11. (Systat Software, San Jose, CA, USA) using a t test for data in Fig. 1A, B, E and F, a two-way ANOVA with repeated measurements for data in Fig. 1C and D, and a two-way ANOVA for the rest of the data. When ANOVAs revealed significant differences, Student Newman Keuls post hoc test was used for multiple comparisons. P <.5 was considered statistically significant. Results AMPKα 2 KO but not AMPKα 1 KO mice have reduced exercise capacity To evaluate metabolism in AMPKα 2 KO mice and littermates as well as in AMPKα 1 KO and their corresponding littermates during exercise at the same relative intensity, mice were initially subjected to a maximum running capacity test on a treadmill. The maximum running speed, achieved by the AMPKα 2 mice, was 38.4 ± 1.2 m min 1, whereas the maximum running speed was reduced by 27% (P <.1) in the AMPKα 2 KO mice (Fig. 1A) as previously published (Jeppesen et al. 213). In contrast, the maximum running speed, achieved by the AMPKα 1 mice, was 32.9 ±.8 m min 1 and was similar in AMPKα 1 KO mice (Fig. 1B). Indirect calorimetry reveals changes in substrate selection in AMPKα 2 KO mice during post-exercise recovery, but not during exercise RER was similar in and AMPKα 2 KO mice and and AMPKα 1 KO mice at rest under fed conditions. This similarity was also found during 2 h of submaximal treadmill exercise at the same relative intensity (Fig. 1C and D, respectively; RER data in the AMPKα 2 KO and mice during running from 18 to 36 min have been published elsewhere (Jeppesen et al. 213)). However, duringthefirst6hofthepost-exerciserecoveryperiod, RER was higher in AMPKα 2 KO mice compared to (P <.5), indicating a lower FA oxidation and a higher glucose oxidation in AMPKα 2 KO mice than in (Fig. 1C and E). Consistently, calculated fatty acid oxidation was lower in AMPKα 2 KO mice than in during the 6 h post-exercise recovery period (Fig. 1G). During the post-exercise recovery period, RER in mice was lower (P <.5) than RER in the resting, non-exercised mice measured at the same circadian hours of the day (from 12. to 18. h), while this was not the case for the AMPKα 2 KO mice. This phenotype was not observed in AMPKα 1 KO mice

6 477 A. M. Fritzen and others J Physiol (Fig. 1D and F) suggesting that AMPK heterotrimeric complexes containing AMPKα 2 rather than AMPKα 1 play a role in the metabolic regulation observed post-exercise. Because a difference in RER during post-exercise recovery was observed only in AMPKα 2 KO mice, the next experiments focused on investigating the mechanisms by which AMPKα 2 regulates substrate selection during post-exercise recovery. previously described (Viollet et al. 23; Jørgensen et al. 24). AMPK Thr 172 (Fig. 2C) andaccser 212 phosphorylation (Fig. 2D) in quadriceps muscle increased (P <.1) 4.2- and 2.9-fold, respectively, in muscle after exercise. This was completely abolished in AMPKα 2 KO muscle. AMPK Thr 172 (Fig. 2C) and ACC Ser 212 phosphorylation (Fig. 2D) 6 h post-exercise were at levels in both genotypes. AMPKα 2 deficient mice have abolished AMPK signalling AMPKα 2 protein expression was ablated in quadriceps muscle of AMPKα 2 KO mice (Fig. 2A), whereas the AMPKα 1 protein expression was higher (P <.1) in the AMPKα 2 KO muscle compared to (Fig. 2B) as Substrate availability during exercise and post-exercise recovery Plasma FA concentration at rest was similar between and AMPKα 2 KO mice, increased (P <.1) with exercise independently of genotype (Table 1) and was similar to levels 6 h post-exercise in both genotypes A C E Maximum speed (m/min) B Maximum speed (m/min) test test AMPKα 2 KO AMPKα 1 KO Respiratory exchange ratio D Respiratory exchange ratio Time (hours) RER during exercise and recovery Time (hours) G FAT utilization (kj/hr/kg) RER during exercise and recovery Fatty acid oxidation during recovery Respiratory exchange ratio Respiratory exchange ratio F Average RER during recovery AMPKα 2 KO Average RER during recovery AMPKα 1 KO Time (hours) Figure 1. AMPKα 2 KO mice are exercise intolerant and display reduced FA oxidation during post-exercise recovery Maximal running speed test in AMPKα 2 KO and (A) andampkα 1 KOandmice(B). Respiratory exchange ratio (RER) during treadmill running at 5% of maximal running speed and in the following 6hpost-exercise recovery period (C and D) and the average RER during 6 h of post-exercise recovery (E and F) inampkα 2 KO and (C and E) andampkα 1 KO and (D and F). G, fatty acid oxidation calculated as described in Methods in AMPKα 2 KO and during 6 h of post-exercise recovery. Data are presented as means ± SEM. n = 8. P <.5, P <.1 significantly different from (main effect of genotype during recovery in C)., wild-type; KO, knock-out.

7 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4771 (Table 1). Plasma TG concentration at rest was similar in the two genotypes and remained unchanged after exercise in, but decreased (P <.5) with exercise in AMPKα 2 KO (Table 1). The plasma TG concentration 6 h post-exercise was at levels in both genotypes (Table 1). Since plasma TG concentration decreased with exercise in AMPKα 2 KO mice, the activity of LPL, which is the rate-limiting enzyme for hydrolysis of plasma lipoprotein-rich TG, was determined in skeletal muscle. LPL activity in gastrocnemius muscle was similar between and AMPKα 2 KO at rest and was unaltered after exercise and 6 h post-exercise in both genotypes (Table 1). This could indicate that the decrease in plasma TG during exercise was probably due to a lower secretion of very low density lipoprotein (VLDL) TG from the liver in AMPKα 2 KO mice rather than altered plasma TG hydrolysis in skeletal muscle. However, since only maximal LPL activity was measured, it cannot be excluded that acute changes in LPL activity such as allosteric or covalent regulation in the capillary bed were responsible fortheobserveddecreaseinplasmatginampkα 2 KO mice. Plasma glucose concentration was similar in and AMPKα 2 KO at rest, and decreased during exercise both in and AMPKα 2 KO (P <.1), but to a greater extent (P <.1) in compared to AMPKα 2 KO (Table 1). Six hours post-exercise plasma glucose concentration was similar to in both genotypes (Table 1). A AMPKα 2 R1 Ex R1 Ex R2 Rec R2 Rec 5 kda AMPKα 2 KO AMPKα 2 KO B AMPKα 1 protein (AU) AMPKα 1 protein. Rest 1 C 5 AMPK phosphorylation pampk Thr 172 (AU) D pacc Ser 212 /ACC protein (AU) Rest 1 ACC phosphorylation E AMPKα 1 pampk pacc ACC Coomassie R1 Ex R1 Ex R2 Rec R2 Rec AMPKα 2 KO AMPKα 2 KO 5 kda 5 kda 25 kda 25 kda Rest 1 Figure 2. Signalling in AMPKα 2 KO and skeletal muscle A,AMPKα 2 protein was absent in AMPKα 2 KO muscle. AMPKα 1 protein content (B), AMPK Thr 172 phosphorylation (C) and ACC Ser 212 phosphorylation/acc protein (D) in quadriceps muscle at rest, immediately after exercise, and 6 h post-exercise recovery. E, representative immunoblots. Data are presented as means ± SEM. n = 7 1. P <.1, P <.1 significantly different from or main effect of genotype. # P <.5 main effect of exercise, ### P <.1 significantly different from rest within. AU, arbitrary units;, wild-type; KO, knock-out. From one litter, and AMPKα 2 KO mice were allocated into a basal, resting group (Rest 1/R1) and an exercise group (Ex). From another litter, and AMPKα 2 KO were allocated into a basal, resting group (/R2) and a recovery group (Rec).

8 4772 A. M. Fritzen and others J Physiol Table 1. Plasma substrates and lipoprotein lipase activity in gastrocnemius muscle at rest, after exercise and after 6 h recovery AMPKα2 KO AMPKα2 KO Rest 1 Rest 1 Plasma glucose (mmol l 1 ) 9.61 ± ±.35 ## 9.55 ± ± 1.27 ##, 8.41 ± ± ± ±.53 Plasma fatty acids (μmol l 1 ) 932 ± ± 188 ### 66 ± ± 183 ### 745 ± ± ± ± 11 Plasma triacyl-glycerol (mmol l 1 ) 1.51 ± ± ± ±.14 # 1.14 ± ± ± ±.13 LPL activity (mu (g wet wt) 1 ) 81. ± ± ± ± ± ± ± ± 5.3 Data are presented as means ± SEM. n = 7 1. ### P <.1 main effect of exercise; # P <.5, ## P <.1 significantly different from rest within genotype; P <.5 main effect of genotype; P <.1 significantly different from within exercise. One milliunit (mu) of LPL activity corresponds to 1 nmol of fatty acid released per minute. From one litter, and AMPKα2 KO mice were allocated into a basal, resting group (Rest 1) and an exercise group. From another litter, and AMPKα2 KO were allocated into a basal, resting group () and a recovery group. Skeletal muscle metabolites during exercise and recovery Glycogen content in the quadriceps muscle was generally higher (P <.1) in compared to AMPKα 2 KO muscle, and was decreased (P <.1) to a similar level in both genotypes with exercise (Fig. 3A). Thus, the relative decrease for the AMPKα 2 KOwaslessthan that for. Six hours post-exercise, muscle glycogen content was restored to a higher (P <.1) level compared to independent of genotype (Fig. 3A). When calculating the difference between the muscle glycogen level after exercise with muscle glycogen level 6 h post-exercise, a lower absolute average muscle glycogen resynthesis rate during the 6 h post-exercise recovery was observed in AMPKα 2 KO muscle (average rate 2.57 mmol (kg wet wt) 1 h 1 ) compared to (average rate 3.36 mmol (kg wet wt) 1 h 1 ). Malonyl-CoA content in the quadriceps muscle was similar in the two genotypes at rest, decreased (P <.1) with exercise independently of genotype and was at levels 6 h post-exercise in both genotypes (Fig. 3B). Free carnitine (Fig. 3C) and cellular free CoA (Fig. 3D) content in quadriceps muscle were similar in and AMPKα 2 KO muscle at rest and decreased (P <.5) after exercise independently of genotype. Six hours post-exercise free carnitine content was at levels in both and AMPKα 2 KO muscle. AMPKα 2 KO mice have blunted exercise-induced PDK4 protein expression and lower NADH content The lower FA oxidation in AMPKα 2 KO compared to mice during post-exercise recovery could not be explained by altered substrate availability. Furthermore, the switch towards an increased carbohydrate oxidation in AMPKα 2 KO rather than FA oxidation as in suggests that the AMPKα 2 KO mice have impaired mitochondrial substrate regulation during post-exercise recovery. Thus, we hypothesized that AMPKα 2 KO muscle displays impaired regulation of the PDH complex. PDK4 protein expression in quadriceps muscle was similar between and AMPKα 2 KO muscle at rest. Interestingly, PDK4 protein expression was increased by 63% with exercise in muscle only (Fig. 4A, P <.1) compared to Rest 1. Six hours post-exercise, PDK4 protein expression was not statistically different from in both genotypes, but tended (P =.6) towards significance in. PDK4 protein was unaltered with exercise and in recovery in AMPKα 2 KO mice (Fig. 4A). PDH-E1α Ser 293 (Fig. 4B) andser 3 phosphorylation (Fig. 4C) in muscle increased (P <.5; P <.1, respectively) with exercise independently of genotype with atrend(p =.9) towards a larger increase in PDH-E1α Ser 293 phosphorylation in mice than in AMPKα 2 KO

9 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4773 mice (Fig. 4B). Six hours post-exercise PDH-E1α Ser 293 and Ser 3 phosphorylation was at levels. The PDH complex is also inhibited by the products NADH and acetyl-coa, both allosterically and by activation of PDK, respectively. Interestingly, NADH content in quadriceps muscle was higher (P <.5) in compared to AMPKα 2 KO at all time points (Fig. 4D) and decreased (P <.1) with exercise, independently of genotype (Fig. 4D). Therefore, AMPKα 2 may influence PDH-E1α by dual mechanisms via up-regulation of PDK4 protein and via inhibition through NADH. Six hours post-exercise the NADH content was similar to levels in both and AMPKα 2 KO (Fig. 4D). Muscle acetyl-coa content (Fig. 4E) and the acetyl-coa/free CoA ratio in muscle (Table 2) were similar in and AMPKα 2 KO at rest and were unaltered immediately after exercise and 6 h post-exercise in both genotypes. Succinyl-CoA content and the NADH/NAD + ratio in the quadriceps muscle were similar in the two genotypes at rest, decreased (P <.1 and P <.1, respectively) with exercise independently of genotype and were similar to levels 6 h post-exercise in both genotypes (Table 2). Similar exercise-induced increase in PDK4 mrna levels, SIRT1 activation and HDAC4 phosphorylation To investigate whether the difference in exercise-induced PDK4 protein expression between genotypes could be related to a transcriptional mechanism, PDK4 gene expression and proposed regulators were assessed. PDK4 mrna level in the quadriceps muscle was similar in and AMPKα 2 KO at rest and increased (P =.5) with exercise independently of genotype (Fig. 5A). Muscle NAD + content was similar in the two genotypes at rest and increased (P <.1) with exercise independently of genotype (Fig. 5B). The similar increase in NAD + content with exercise in the two genotypes was accompanied by a similar decrease (P <.5) in p53 Lys 379 acetylation in quadriceps muscle (Fig. 5C) implying similar SIRT1 activation with exercise in and AMPKα 2 KO muscle. HDAC4 Ser 632 phosphorylation in quadriceps was similar in the two genotypes at rest and decreased (P <.5) with exercise independently of genotype (Fig. 5D). To examine whether the difference in exercise-induced PDK4 protein expression between genotypes could be related to a difference in translational regulation, the content of mir-17, a proposed regulator of PDK4 translation, was measured. However, mir-17 level did not change with exercise and was not different between genotypes (Fig. 5E). RNU6B, snorna135, snorna22 and snorna234 mrna levels were determined as potential endogenous controls, but they all changed with either exercise or genotype. Thus, mir-17 is not shown as normalized to any endogenous control. Even so, when mir-17 level was related to each of the investigated controls, mir-17 still did not change with exercise and was not different between genotypes, thus A Muscle glycogen content (mmol/kg w.w.) C Free carnitine (mmol/kg. d.w.) Rest 1 Muscle glycogen Free carnitine B Free CoA (nmol/mg w.w.) Malonyl-CoA (nmol/mg w.w.) D Rest 1 Malonyl-CoA Free CoA. Rest 1 Rest 1 Figure 3. Content of glycogen (A), malonyl-coa (B), free carnitine (C) and free CoA (D) in quadriceps muscle at rest, immediately after exercise, and 6 h post-exercise recovery Data are presented as means ± SEM. n = 7 1. # P <.5, ### P <.1 main effect of exercise. P <.1 main effect of genotype. w.w., wet weight; d.w., dry weight. From one litter, and AMPKα 2 KO mice were allocated into a basal, resting group (Rest 1) and an exercise group. From another litter, and AMPKα 2 KO were allocated into a basal, resting group () and a recovery group.

10 4774 A. M. Fritzen and others J Physiol underlining no changes in mir-17 with exercise in either genotype. Discussion Previous studies have shown that FA oxidation is elevated several hours post-exercise in humans, occurring concomitantly with a restoration of muscle glycogen stores (Wolfe et al. 199; Melby et al. 1993; Kiens & Richter, 1998), even when a carbohydrate-rich diet is consumed (Kiens & Richter, 1998). The mechanisms regulating this selection of energy substrate towards oxidation/resynthesis in skeletal muscle during the recovery from exercise have remained unsolved. It is well known that AMPK deficiency decreases exercise tolerance in mice (Maarbjerg et al. 29; O Neill et al. 211; Jeppesen et al. 213; Fentzet al. 215). Therefore, we chose to let mice lacking AMPKα 2 and littermates exercise at the same relative intensity thereby probably stressing the muscle equally between genotypes during exercise. Accordingly, a greater total amount of exercise at a higher absolute rate of O 2 consumption ( V O2 ) was performed by resulting in a greater use of muscle glycogen and plasma glucose during the exercise bout. Despite this, we here demonstrate an equal high relative reliance in both genotypes on FA oxidation for energy production (covering about 8% of energy production) during prolonged and glycogen-depleting exercise. However, immediately post-exercise AMPKα 2 KO mice displayed a markedly suppressed FA utilization compared to. This phenotype in substrate selection continued for several hours during recovery from exercise and suggests a role for AMPK in regulation of substrate utilization during recovery. We propose that A 2. PDK4 protein D 1 NADH PDK4 protein (AU) NADH (nmol/mg d.w.) B ppdh Ser /PDH-E1α (AU) Rest 1 PDH phosphorylation E Acetyl-CoA (nmol/mg w.w.) Rest 1 Acetyl-CoA C ppdh Ser /PDH-E1α (AU) Rest 1 PDH phosphorylation Rest 1 F PDK4 ppdh Ser 293 ppdh Ser 3 PDH-E1α Rest 1 R1 Ex R1 Ex R2 Rec R2 Rec 37 kda 37 kda 37 kda 37 kda Coomassie AMPKα 2 KO AMPKα 2 KO Figure 4. AMPKα 2 KO mice have blunted exercise-induced PDK4 protein expression and lower NADH content in muscle PDK4 protein expression (A), PDH-E1α Ser 293 phosphorylation/pdh-e1α protein (B), PDH-E1α Ser 3 phosphorylation/pdh-e1α protein (C), NADH content (D) and acetyl-coa content (E) in quadriceps muscle at rest, immediately after exercise, and 6 h post-exercise recovery. F, representative immunoblots. Data are presented as means ± SEM. n = 7 1. ## P <.1, ### P <.1 main effect of exercise (D) and significantly different from rest within (A); P <.5, P <.1 main effect of genotype; P <.1 significantly different from within exercise. AU, arbitrary units. From one litter, and AMPKα 2 KO mice were allocated into a basal, resting group (Rest 1/R1) and an exercise group (Ex). From another litter, and AMPKα 2 KO were allocated into a basal, resting group (/R2) and a recovery group (Rec).

11 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4775 Table 2. Metabolites in the quadriceps muscle at rest, after exercise and after 6 h recovery AMPKα2 KO AMPKα2 KO Rest 1 Rest 1 Succinyl-CoA (mmol (mg wet wt) 1 ) 8.7 ± ±.66 ## 7.54 ± ±.2 ## 7.24 ± ± ± ±.31 Acetyl CoA/free CoA.28 ±.1.29 ±.3.31 ±.2.28 ±.2.33 ±.2.32 ±.1.3 ±.1.33 ±.4 NADH/NAD ± ±.14 ### 1.68 ± ±.14 ### 2.62 ± ± ± ±.34 Data are presented as means ± SEM. n = 7 1. ## P <.1, ### P <.1 main effect of exercise. From one litter, and AMPKα2 KO mice were allocated into a basal, resting group (Rest 1) and an exercise group. From another litter, and AMPKα2 KO were allocated into a basal, resting group () and a recovery group. AMPK promotes lipid oxidation in the recovery period by up-regulation of PDK4 expression, stimulated by prior exercise (during exercise) in turn phosphorylating and inhibiting PDH activity in muscle post-exercise. This would lead to a lower glucose oxidation, which would indirectly drive a demand for an increased FA oxidation. In addition, AMPK deficiency led to lower muscle concentrations of NADH, which is a known activator of PDK4. PDK4 is an important enzyme in the regulation of glucose consumption in skeletal muscle (Rowles et al. 1996; Bowker-Kinley et al. 1998). PDK4 inhibits PDH activity by phosphorylation (Linn et al. 1969), thereby inhibiting the rate-limiting multi-enzyme complex responsible for the irreversible decarboxylation of pyruvate to acetyl-coa. Recently, an impaired PDH regulation was observed in PDK4 whole-body KO mice during acute muscle contractions ex vivo (Herbst et al. 212) indicating an important role of PDK4 in metabolic regulation during exercise. In the same mice, a 5% decreased liver but not muscle glycogen synthesis rate was observed in the hours after exercise when pair-fed (Herbst et al. 214) suggesting that PDK4 may not be involved in the regulation of muscle glycogen repletion post-exercise. However, when not pair-fed, caloric consumption was doubled by PDK4 KO mice compared to during post-exercise recovery (Herbst et al. 214), which in itself indicates an importance of PDK4 during post-exercise recovery. Moreover, the and PDK4 KO mice in that study were not littermates and were bred in different facilities making a clear interpretation of these findings difficult. In the present study, exercise increased the PDK4 protein content in muscle from mice, but interestingly not in AMPKα 2 deficient mice. This finding was further supported by the observation of an increased phosphorylation of PDH-E1α at Ser 293 during exercise in mice, but not AMPKα 2 KO muscle. Previously it was shown in pig heart that PDK4 was activated by NADH, in turn inactivating PDH (Garland, 1964; Linn et al. 1969; Cooper et al. 1975; Ravindran et al. 1996). Here, we show that AMPKα 2 deficient muscles generally have reduced NADH content strengthening the point that AMPKα 2 KO mice have impaired suppression of PDH activity. However, the total NADH content was measured in whole muscle tissue, and it is only the mitochondrial NADH that affects PDH activity. This, however, represents by far the biggest part of the total NADH content (Sahlin & Katz, 1986; Dash et al. 28; Li et al. 29; White & Schenk, 212). The mechanism by which AMPK activation during exercise regulates PDK4 protein content in skeletal muscle could be in multiple steps from transcription to translation in the synthesis process of new protein as well as stability of the protein. In the present study, PDK4 mrna was shown to increase (P =.5) after a single

12 4776 A. M. Fritzen and others J Physiol bout of exercise in both AMPKα 2 KO and muscle. This is in line with previous findings after both a single AICAR injection and treadmill exercise in the same mouse model (Jørgensen et al. 25). Together these findings clearly demonstrate that defective AMPKα 2 does not robustly affect exercise-induced gene expression of PDK4. However, mrna levels do not necessarily reflect transcriptional activity. To further investigate exercise-induced PDK4 transcription, the activation of the two major regulators of transcription, SIRT1 and HDAC4, inducing PDK4 transcription (Fulco et al. 23; Furuyama et al. 23; Brunet et al. 24; Gerhart-Hines et al. 27; Cantó et al. 29; Mihaylova et al. 211), were studied. induced a similar increase in NAD + content and a decrease in acetylation of p53, implying a similar activation of the NAD + dependent deacetylase SIRT1 in both genotypes. Likewise, exercise decreased HDAC4 phosphorylation similarly in the two genotypes, which is expected to result in a similar exclusion of HDAC4 from the nucleus, thereby activating the FoxO1 transcription factor (Mihaylova et al. 211) and accordingly inducing PDK4 transcription. Collectively, this supports the finding of a similar exercise-induced increase in PDK4 mrna in the two genotypes. The lack of an effect of AMPKα 2 depletion on exercise-induced SIRT1 activation is in contrast to studies in C2C12 myotubes transfected with a retrovirus expressing a dominant negative form of AMPK during glucose restriction (Fulco et al. 28) and mouse muscle deficient in AMPKγ 3 performing swimming exercise (Cantó et al. 21) showingampk to be necessary for activation of SIRT1. These discrepancies are difficult to explain, but could be due to compensatory up-regulation of AMPKα 1 protein expression in AMPKα 2 KO muscle (Jørgensen et al. 25, 27) or more likely a difference in the response to different exercise regimes. The robust finding of an increased PDK4 protein expression in muscle of mice already after 2 h of exercise is striking. Previous studies have shown that 4 9 min of treadmill running in mice (Jørgensen et al. 25; Cantó et al. 29; Kiilerich et al. 21) and6 15minofexerciseinhumans (Pilegaard et al. 2, 22, 25) induced a 2- to 8-fold increase in PDK4 transcriptional activity and/or mrna level in muscle immediately after or in the hours after exercise. It seems that the prolonged, exhaustive exercise bout with a depletion of energy stores and high substrate flux in mice has been the foundation for the increase in PDK4 protein within just 2 h of exercise. The exercise-induced increase in PDK4 protein expression observed only in mice, despite similar PDK4 mrna levels between and AMPKα 2 KO muscle, point towards a role of AMPK in post-transcriptional regulation of PDK4 protein. Recently, A PDK4 mrna B NAD + C phdac4 Ser 632 /HDAC4 (AU) PDK4 mrna/ssdna P=.5 D HDAC4 phosphorylation E mir-17 F Rest Rest Rest NAD + content (nmol/mg d.w.) mir-17 (AU) Rest Acetylation p53 Lys 379 /p53 protein (AU) a-p53 p53 phdac4 HDAC4 Rest p53 acetylation Re Ex Re Ex AMPKα 2 KO 5 kda 5 kda 1 kda 1 kda Figure 5. Similar exercise-induced increase in PDK4 mrna levels, p53 acetylation and decreased HDAC4 phosphorylation in AMPKα 2 KO and muscle PDK4 mrna levels (A), NAD + content (B), p53 Lys 379 acetylation/p53 protein (C), HDAC4 Ser 632 phosphorylation/hdac4 protein (D), and mir-17 gene expression (E) in quadriceps muscle at rest and immediately after 2 h of treadmill exercise. F, representative immunoblots. Data are presented as means ± SEM. n = 7 1. # P <.5, ## P <.1, ### P <.1 main effect of exercise. AU, arbitrary units; Ex, exercise; d.w., dry weight; Re, rest.

13 J Physiol AMPK controls substrate metabolism during post-exercise recovery 4777 AMPK activation was shown to suppress endothelial cell expression of angiotensin converting enzyme post-translationally by phosphorylation of p53 and up-regulation of microrna (mir) 143/145 (Kohlstedt et al. 213) suggesting a role for AMPK in translational regulation through mir-mediated regulation. mir-17 was predicted to regulate PDK4 translation (Wilfred et al. 27) and suggested to be important in the regulation of PDK4 protein content after exercise in mouse muscle (Safdar et al. 29). However, the observation that mir-17 expression was independent of genotype and not affected by exercise suggests that AMPKα 2 does not regulate the protein abundance of PDK4 via a mir-17-induced regulation. The observed genotypic difference in substrate selection during post-exercise recovery could not be explained by differences in circulating FA and TG concentrations or lipoprotein lipase activity in skeletal muscle. Furthermore, muscle malonyl-coa levels, which have been associated with enhanced FA oxidation during post-exercise recovery in rat skeletal muscle (Rasmussen et al. 1998), cannot explain the difference in FA oxidation during recovery in the present study, as malonyl-coa content was similar between and AMPKα 2 KO muscle both immediately after exercise and 6 h post-exercise. The level of free carnitine and free CoA availability, which are both involved in the conversion of FAs into fatty acylcarnitines (van Loon et al. 21; Roepstorff et al. 25; Jeppesen et al. 213), the form required for mitochondrial FA transmembrane transport, did not differ between genotypes. Taken together this suggests that AMPK is critical for mitochondrial substrate selection towards FA oxidation in the post-exercise period through regulation of the PDH complex. Importantly, the high FA oxidation during Thr172 AMP NADH AMPK α 2 γ β PDK4 PDH GLU OX pyruvate acetyl-coa FA OX Figure 6. Scheme of proposed AMPK-mediated regulation of fuel selection during post-exercise recovery -induced increase in AMPKα 2 activity in skeletal muscle increases pyruvate dehydrogenase 4 (PDK4) protein content, and AMPKα 2 seems also to be crucial for NADH levels. Both inhibit pyruvate dehydrogenase (PDH) activity, whereby conversion of pyruvate to acetyl-coa is inhibited and consequently glucose oxidation (GLU OX). This enables increased fatty acid oxidation (FA OX). recovery enables resynthesis of glucose towards muscle glycogen rather than oxidation as shown by calculated lower average muscle glycogen storage post-exercise in AMPKα 2 KO mice. This is in line with earlier observations showing lower muscle glycogen levels during the post-exercise recovery period in muscle lacking functional AMPKα 2 (Mu et al. 23; Jørgensen et al. 25) or AMPKγ 3 (Barnes et al. 24), although different recovery time periods have been examined. A potential limitation in the study could be that food intake during the post-exercise recovery period was not measured. However, it has previously been observed that food intake in AMPKα 2 KO mice is similar to (Viollet et al. 23). Moreover, in the present study, mice utilized a greater amount of energy and muscle glycogen during the exercise bout at the same relative intensity as AMPKα 2 KOmice.Ifanythingthiswouldbeexpected to lead to a higher intake of carbohydrate-rich chow food, resulting in a higher RER in post-exercise. But the opposite was observed. Thus, taken together, it seems unlikely that potential differences in food intake post-exercise can explain the observed findings in the present study. In conclusion, we here propose a mechanism responsible for the established observation of a higher FA oxidation during post-exercise recovery in favour of resynthesis of muscle glycogen stores (see Fig. 6). Thus, the present findings demonstrate that AMPKα 2 plays an important, but indirect role, in increasing FA oxidation in muscle during post-exercise recovery. This seems to occur through an AMPKα 2 PDK4-mediated inhibition of the mitochondrial PDH complex, and not through a reduction in muscle malonyl-coa levels or changes in free carnitine or CoA availability. The inhibition of PDH activity decreases carbohydrate oxidation creating a demand for FA oxidation following exercise. Consequently, the glucose taken by muscle is directed primarily for glycogen synthesis rather than oxidation. References Barnes BR, Marklund S, Steiler TL, Walter M, Hjalm G, Amarger V, Mahlapuu M, Leng Y, Johansson C, Galuska D, Lindgren K, Abrink M, Stapleton D, Zierath JR & Andersson L (24). The 5 -AMP-activated protein kinase γ3isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 279, Bielinski R, Schutz Y & Jequier E (1985). Energy metabolism during the postexercise recovery in man. Am J Clin Nutr 42, Bowker-Kinley MM, Davis WI, Wu P, Harris RA & Popov KM (1998). Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 329,