ampk e regulação do metabolismo

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J Physiol 593.21 (2015) pp 4765–4780 4765
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5′-AMP activated protein kinase α2 controls substrate
metabolism during post-exercise recovery via regulation
of pyruvate dehydrogenase kinase 4
Andreas Mæchel Fritzen1, Anne-Marie Lundsgaard1,4, Jacob Jeppesen1,3,
Mette Landau Brabæk Christiansen1, Rasmus Biensø2, Jason R. B. Dyck5, Henriette Pilegaard2
and Bente Kiens1
1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, the August Krogh Centre, University of Copenhagen, Copenhagen,
Denmark
2Centre of Inflammation and Metabolism, the August Krogh Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark
3Type 2 Diabetes and Obesity Pharmacology, Novo Nordisk A/S, Maaloev, Denmark
4Danish Diabetes Academy, Odense University Hospital, Odense, Denmark
5Cardiovascular 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 α2 (AMPKα2).
� AMPKα2 is involved in post-transcriptional and not transcriptional regulation of pyruvate
dehydrogenase kinase 4 (PDK4) in muscle.
� Exercise-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, sub-
strate oxidation was measured after prolonged exercise and during the following 6 h post-exercise
in 5´-AMP activated protein kinase (AMPK) α2 and α1 knock-out (KO) and wild-type (WT)
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 < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange
ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without
genotype differences in muscle malonyl-CoA or free-carnitine concentrations. A similar increase
in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO
was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the
exercise-induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Inter-
estingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in
AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01)
inhibitory pyruvate dehydrogenase (PDH)-E1α Ser293 phosphorylation was observed in AMPKα2
KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism
post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently
creating conditions for increased fatty acid oxidation.
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society DOI: 10.1113/JP270821
4766 A. M. Fritzen and others J Physiol 593.21
(Resubmitted 15 June 2015; accepted after revision 2 September 2015; first published online 11 September 2015)
Corresponding author B. Kiens: Department of Nutrition, Exercise and Sports, the August Krogh Centre, University of
Copenhagen, Universitetsparken 13, 2100 Copenhagen Ø, Denmark. Email: bkiens@nexs.ku.dk
Abbreviations ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide riboside; AMPK, 5′-AMP
activated protein kinase; CT, 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; WT, 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. 1990; Wolfe et al. 1990;
Melby et al. 1993; Calles-Escandon et al. 1996; Treuth
et al. 1996; Horton et al. 1998; Kiens & Richter, 1998;
Kimber et al. 2003). Even with an increased dietary 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. 2003). 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. 2003). 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
(α1 or α2) in combination with a regulatory β- (β1 or
β2) and γ-subunit (γ1, γ2 or γ3), is activated by increased
AMP:ATP and ADP:ATP ratios and stimulates substrate
metabolism (Hardie, 2008; Richter & Ruderman, 2009).
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 peri-
ods 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. (2007) showed an increased PDH activity
in skeletal muscle from AMPKα2 knock-out (KO) mice
compared with wild-type (WT) at rest and during exercise
(Klein et al. 2007). A key enzyme in controlling the activity
of PDH is pyruvate dehydrogenase kinase 4 (PDK4),
which acts by inhibition of PDH activity and thereby pre-
venting 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. 2009) 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. 2009; Cantó &
Auwerx, 2011), and studies in human muscle samples
suggested an association between AMPK activity and
HDAC4 export from the nucleus (McGee et al. 2009).
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 thehours following prolonged exercise (Kimber
et al. 2003). 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 30–90 min of
treadmill running or 2 h of swimming, were lower in mice
lacking functional AMPK than in WT (Mu et al. 2003;
Barnes et al. 2004; Jørgensen et al. 2005).
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 α1 and α2
AMPK-containing complexes are present in skeletal
muscle and potentially could regulate fuel selection
post-exercise, we investigated both AMPKα1 and AMPKα2
KO mice and their respective WT littermates in metabolic
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
J Physiol 593.21 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 Inspec-
torate 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 AMPKα1
or AMPKα2 containing heterotrimers, female AMPKα1
and AMPKα2 whole-body KO and their respective WT
littermates were used. Mice were 12–16 weeks during
the characterization experiments and 17–21 weeks at
their final termination. The mice were generated as pre-
viously described (Viollet et al. 2003; Jørgensen et al.
2004). Briefly, AMPKα1 KO mice were generated on
129sv background, and AMPKα2 KO mice were generated
on C57Bl/6 background. In both models, homozygote
WT and KO littermates were generated by heterozygote
inter-cross breeding. AMPKα2 KO mice have a normal
lifespan with body weight, body composition and food
intake similar to WT (Viollet et al. 2003). Genotyping
was performed by PCR analysis as previously described
(Jørgensen et al. 2004; Thomson et al. 2007) 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 0% 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, 10 min at 17 m min−1. Thereafter all
mice performed a maximal running speed test, in which
the mice started at 10.8 m min−1 at a 0% 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 CO2 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
CO2 production/O2 uptake. FAT utilization was calculated
as 19 kJ/l O2 ∗ oxygen uptake (l O2/h/kg)·(100% -
((RER-0.7)/0.3)).
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, AMPKα1 KO and WT littermates as well
as AMPKα2 KO and WT littermates underwent 2 h of
treadmill exercise at 50% of average maximal running
speed per group. AMPKα1 KO and WT mice ran at
16 m min−1, and AMPKα2 KO and WT 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, and O2 uptake
and CO2 production were measured during the following
6 h post-exercise recovery period.
Thereafter, WT and AMPKα2 KO mice were allocated
into a basal, resting group (Rest 1: WT, n = 10; KO, n = 10)
and an exercise group (WT, n = 10; KO, n = 10). Another
group of WT and AMPKα2 KO mice were allocated
into a basal, resting group (Rest 2: WT, n = 9; KO,
n = 8) and a recovery group (WT, n = 10; 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 50% of individual group maximal running
speed between 16.00 and 18.00 h. The recovery group
performed initially 2 h of treadmill exercise at 50% of
individual group maximal running speed between 10.00
and 12.00 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 Rest 2) had free access to
water and food until termination. All mice were killed
by cervical dislocation at 18.00 h, which was the same
standardized time of the day for all animals. Gastro-
cnemius and quadriceps muscles were quickly removed
and immediately frozen in liquid nitrogen and stored at
−80°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
30 μl of 200 mM EGTA. The blood was centrifuged in
a Dich centrifuge at 18,000 g (type 155SLRC-BK; Ole
Dich Instrumentmakers, Denmark) for 3 min, after which
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
4768 A. M. Fritzen and others J Physiol 593.21
plasma was collected and stored at −20°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 AMPKα2 as well as in
WT mice (Mu et al. 2003), 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 340 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 10–15 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.
1990) as described previously (Roepstorff et al. 2005).
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 �20 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. 2000). 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. 2000). The cDNA samples
were diluted to a concentration of 60 μl μg−1 mRNA
in nuclease-free H2O. 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. (2006). 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-200; MJ Research, Waltham, MA, USA).
Real time PCR was performed with an ABI 7900
sequence-detection system (Applied Biosystems) and has
previously been published (Kiilerich et al. 2010). Primers
and TaqMan probes for amplifying a PDK4-specific
mRNA fragment were designed using the mouse-specific
database from Ensembl (http://www.ensembl.org/index.
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 (CT) 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-107, RNU6B, snoRNA135, snoRNA202
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 CT 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 (50 mg wet wt) were incubated in a [3H]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 [3H]oleic acid during the last 60 min of the
110 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
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
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J Physiol 593.21 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 micro-
scope, and �5 mg dry wt was homogenized in ice-cold
buffer as previously described (Jeppesen et al. 2010)
modified with deacetylase inhibition by adding 10 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. 23227; 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. P0397: Dako,
Glostrup, Denmark), anti-AMPKα1 and anti-AMPKα2
(kindly donated by Dr Hardie, Dundee University,
UK). The following primary phospho-specific anti-
bodies were used: anti-ACC Ser79 phosphorylation (no.
07-303, 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 Ser79 phosphorylation site,
but the antibody also recognises the mouse ACC2
when phosphorylated at the corresponding Ser212), and
anti-α-AMPK Thr172 phosphorylation (no. 2535, Cell
Signaling Technology, Danvers, MA, USA). Furthermore
anti-p53 (no. 2524) and anti-acetylated p53 Lys379
(no. 2570) (Cell Signaling Technology) were used.
In addition, protein levels of the PDH-E1α sub-
unit, PDH-E1α phosphorylation at Ser293 and Ser300
(Pilegaard et al. 2006) and pyruvate dehydrogenase
kinase isozyme 4 (PDK4) protein levels (Kiilerich et al.
2010) 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 4.0, BioRad) and expressed as arbitrary
units. Membranes used for detection of phosphorylated
ACC and acetylated p53 were stripped with a buffer
containing 100 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 10 min in 0.5% Coomassie
Blue G-250 in 50% ethanol–10% acetic acid, removal of
excess Coomassie stain with distilled water followed by
destaining in 50% ethanol–10% acetic acid until bands
were clearly visible.
Statistics
Data are expressed as means ± SEM. All statistical analyses
were performed in SigmaPlot 11.0 (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 < 0.05 was considered
statistically significant.
Results
AMPKα2 KO but not AMPKα1 KO mice have reduced
exercise capacity
To evaluate metabolism in AMPKα2 KO mice and
WT littermates as well as in AMPKα1 KO and their
corresponding WT 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 AMPKα2 WT
mice, was 38.4 ± 1.2 m min−1, whereas the maximum
running speed was reduced by 27% (P < 0.001) in the
AMPKα2 KO mice (Fig. 1A) as previously published
(Jeppesen et al. 2013). In contrast, the maximum
running speed, achieved by the AMPKα1 WT mice, was
32.9 ± 0.8 m min−1 and was similar in AMPKα1 KO mice
(Fig. 1B).
Indirect calorimetry reveals changes in substrate
selection in AMPKα2 KO mice during post-exercise
recovery, but not during exercise
RER was similar in WT and AMPKα2 KO mice and
WT and AMPKα1 KO mice at rest under fed conditions.
This similarity was alsofound during 2 h of submaximal
treadmill exercise at the same relative intensity (Fig. 1C
and D, respectively; RER data in the AMPKα2 KO and
WT mice during running from 18 to 36 min have been
published elsewhere (Jeppesen et al. 2013)). However,
during the first 6 h of the post-exercise recovery period,
RER was higher in AMPKα2 KO mice compared to
WT (P < 0.05), indicating a lower FA oxidation and
a higher glucose oxidation in AMPKα2 KO mice than
in WT (Fig. 1C and E). Consistently, calculated fatty
acid oxidation was lower in AMPKα2 KO mice than
in WT during the 6 h post-exercise recovery period
(Fig. 1G). During the post-exercise recovery period,
RER in WT mice was lower (P < 0.05) than RER in
the resting, non-exercised mice measured at the same
circadian hours of the day (from 12.00 to 18.00 h),
while this was not the case for the AMPKα2 KO mice.
This phenotype was not observed in AMPKα1 KO mice
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
4770 A. M. Fritzen and others J Physiol 593.21
(Fig. 1D and F) suggesting that AMPK heterotrimeric
complexes containing AMPKα2 rather than AMPKα1 play
a role in the metabolic regulation observed post-exercise.
Because a difference in RER during post-exercise recovery
was observed only in AMPKα2 KO mice, the next
experiments focused on investigating the mechanisms
by which AMPKα2 regulates substrate selection during
post-exercise recovery.
AMPKα2 deficient mice have abolished AMPK
signalling
AMPKα2 protein expression was ablated in quadriceps
muscle of AMPKα2 KO mice (Fig. 2A), whereas the
AMPKα1 protein expression was higher (P < 0.001) in
the AMPKα2 KO muscle compared to WT (Fig. 2B) as
previously described (Viollet et al. 2003; Jørgensen
et al. 2004). AMPK Thr172 (Fig. 2C) and ACC Ser212
phosphorylation (Fig. 2D) in quadriceps muscle increased
(P < 0.001) 4.2- and 2.9-fold, respectively, in WT muscle
after exercise. This was completely abolished in AMPKα2
KO muscle. AMPK Thr172 (Fig. 2C) and ACC Ser212
phosphorylation (Fig. 2D) 6 h post-exercise were at Rest 2
levels in both genotypes.
Substrate availability during exercise and
post-exercise recovery
Plasma FA concentration at rest was similar between
WT and AMPKα2 KO mice, increased (P < 0.001) with
exercise independently of genotype (Table 1) and was
similar to Rest 2 levels 6 h post-exercise in both genotypes
50
A C E
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Time (hours)
Time (hours)
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Exercise test RER during exercise and recovery
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20
10
0
Figure 1. AMPKα2 KO mice are exercise intolerant and display reduced FA oxidation during post-exercise
recovery
Maximal running speed test in AMPKα2 KO and WT (A) and AMPKα1 KO and WT mice (B). Respiratory exchange
ratio (RER) during treadmill running at 50% of maximal running speed and in the following 6 h post-exercise
recovery period (C and D) and the average RER during 6 h of post-exercise recovery (E and F) in AMPKα2 KO and
WT (C and E) and AMPKα1 KO and WT (D and F). G, fatty acid oxidation calculated as described in Methods in
AMPKα2 KO and WT during 6 h of post-exercise recovery. Data are presented as means ± SEM. n = 8. ∗P < 0.05,
∗∗∗P < 0.001 significantly different from WT (main effect of genotype during recovery in C). WT, wild-type; KO,
knock-out.
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
J Physiol 593.21 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 WT, but decreased (P < 0.05) with exercise
in AMPKα2 KO (Table 1). The plasma TG concentration
6 h post-exercise was at Rest 2 levels in both genotypes
(Table 1). Since plasma TG concentration decreased with
exercise in AMPKα2 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
WT and AMPKα2 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 AMPKα2 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
for the observed decrease in plasma TG in AMPKα2 KO
mice. Plasma glucose concentration was similar in WT
and AMPKα2 KO at rest, and decreased during exercise
both in WT and AMPKα2 KO (P < 0.01), but to a
greater extent (P < 0.01) in WT compared to AMPKα2
KO (Table 1). Six hours post-exercise plasma glucose
concentration was similar to Rest 2 in both genotypes
(Table 1).
2.5
R1
A
C
D
E
B
WTAMPKα2KO
AMPKα1 protein
AMPKα2
AMPKα1
AMPKα2KO AMPKα2KO
pAMPK
pACC
ACC
Coomassie
WT WT
WTAMPKα2KO
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
R1 R2 R2Rec Rec
50 kDa
50 kDa
50 kDa
250 kDa
250 kDa
RecoveryRest 2Rest 1
5
4
3
2
1
0
4
3
2
1
0
Exercise
AMPK phosphorylation
ACC phosphorylation
RecoveryRest 2Rest 1 Exercise
Ex Ex
R1 R1 R2 R2Rec RecEx Ex
2.0
A
M
P
K
α
1
 p
ro
te
in
 (
A
U
)
p
A
M
P
K
 T
h
r1
7
2
 (
A
U
)
p
A
C
C
 S
e
r2
1
2
/A
C
C
 p
ro
te
in
 (
A
U
)
1.5
1.0
0.5
0.0
Rest 1 Rest 2Exercise Recovery
Figure 2. Signalling in AMPKα2 KO and WT skeletal muscle
A, AMPKα2 protein was absent in AMPKα2 KO muscle. AMPKα1 protein content (B), AMPK Thr172 phosphorylation
(C) and ACC Ser212 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 = 7–10.
∗∗P < 0.01, ∗∗∗P < 0.001 significantly different from WT or main effect of genotype. #P < 0.05 main effect of
exercise, ###P < 0.001 significantly different from rest within WT. AU, arbitrary units; WT, wild-type; KO, knock-out.
From one litter, WT and AMPKα2 KO mice were allocated into a basal, resting group (Rest 1/R1) and an exercise
group (Ex). From another litter, WT and AMPKα2 KO were allocated into a basal, resting group (Rest 2/R2) and a
recovery group (Rec).
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
4772 A. M. Fritzen and others J Physiol 593.21
Ta
b
le
1.
Pl
as
m
a
su
b
st
ra
te
s
an
d
lip
o
p
ro
te
in
lip
as
e
ac
ti
vi
ty
in
g
as
tr
o
cn
em
iu
s
m
u
sc
le
at
re
st
,a
ft
er
ex
er
ci
se
an
d
af
te
r
6
h
re
co
ve
ry
W
T
A
M
PK
α
2
K
O
W
T
A
M
PK
α
2
K
O
R
es
t
1
Ex
er
ci
se
R
es
t
1
Ex
er
ci
se
R
es
t
2
R
ec
o
ve
ry
R
es
t
2
R
ec
o
ve
ry
Pl
as
m
a
g
lu
co
se
(m
m
o
ll
−1
)
9.
61
±
0.
45
3.
13
±
0.
35
##
9.
55
±
0.
42
6.
30
±
1.
27
##
,∗
∗
8.
41
±
0.
37
8.
15
±
0.
28
8.
93
±
0.
71
∗
9.
88
±
0.
53
∗
Pl
as
m
a
fa
tt
y
ac
id
s
(μ
m
o
ll
−1
)
93
2
±24
8
16
02
±
18
8#
##
66
0
±
93
14
21
±
18
3#
##
74
5
±
90
83
2
±
71
83
6
±
92
82
0
±
11
0
Pl
as
m
a
tr
ia
cy
l-
g
ly
ce
ro
l(
m
m
o
ll
−1
)
1.
51
±
0.
10
1.
60
±
0.
12
1.
74
±
0.
03
1.
40
±
0.
14
#
1.
14
±
0.
08
1.
28
±
0.
13
1.
26
±
0.
17
1.
37
±
0.
13
LP
L
ac
ti
vi
ty
(m
U
(g
w
et
w
t)
−1
)
81
.0
±
3.
4
84
.8
±
2.
9
79
.8
±
3.
9
79
.6
±
4.
3
13
0
±
13
14
1
±
4.
6
14
6
±
6.
3
15
4
±
5.
3
D
at
a
ar
e
p
re
se
n
te
d
as
m
ea
n
s
±
SE
M
.n
=
7–
10
.#
##
P
<
0.
00
1
m
ai
n
ef
fe
ct
o
f
ex
er
ci
se
;#
P
<
0.
05
,#
#
P
<
0.
01
si
g
n
ifi
ca
n
tl
y
d
if
fe
re
n
t
fr
o
m
re
st
w
it
h
in
g
en
o
ty
p
e;
∗ P
<
0.
05
m
ai
n
ef
fe
ct
o
f
g
en
o
ty
p
e;
∗∗
P
<
0.
01
si
g
n
ifi
ca
n
tl
y
d
if
fe
re
n
t
fr
o
m
W
T
w
it
h
in
ex
er
ci
se
.
O
n
e
m
ill
iu
n
it
(m
U
)
o
f
LP
L
ac
ti
vi
ty
co
rr
es
p
o
n
d
s
to
1
n
m
o
l
o
f
fa
tt
y
ac
id
re
le
as
ed
p
er
m
in
u
te
.
Fr
o
m
o
n
e
lit
te
r,
W
T
an
d
A
M
PK
α
2
K
O
m
ic
e
w
er
e
al
lo
ca
te
d
in
to
a
b
as
al
,r
es
ti
n
g
g
ro
u
p
(R
es
t
1)
an
d
an
ex
er
ci
se
g
ro
u
p
.F
ro
m
an
o
th
er
lit
te
r,
W
T
an
d
A
M
PK
α
2
K
O
w
er
e
al
lo
ca
te
d
in
to
a
b
as
al
,
re
st
in
g
g
ro
u
p
(R
es
t
2)
an
d
a
re
co
ve
ry
g
ro
u
p
.
Skeletal muscle metabolites during exercise
and recovery
Glycogen content in the quadriceps muscle was generally
higher (P < 0.001) in WT compared to AMPKα2 KO
muscle, and was decreased (P < 0.001) to a similar
level in both genotypes with exercise (Fig. 3A). Thus,
the relative decrease for the AMPKα2 KO was less than
that for WT. Six hours post-exercise, muscle glycogen
content was restored to a higher (P < 0.001) level
compared to Rest 2 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 AMPKα2 KO muscle (average rate 2.57 mmol
(kg wet wt)−1 h−1) compared to WT (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 < 0.01) with exercise independently
of genotype and was at Rest 2 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 WT and AMPKα2 KO muscle at rest
and decreased (P < 0.05) after exercise independently of
genotype. Six hours post-exercise free carnitine content
was at Rest 2 levels in both WT and AMPKα2 KO
muscle.
AMPKα2 KO mice have blunted exercise-induced
PDK4 protein expression and lower NADH content
The lower FA oxidation in AMPKα2 KO compared to
WT mice during post-exercise recovery could not be
explained by altered substrate availability. Furthermore,
the switch towards an increased carbohydrate oxidation
in AMPKα2 KO rather than FA oxidation as in WT
suggests that the AMPKα2 KO mice have impaired
mitochondrial substrate regulation during post-exercise
recovery. Thus, we hypothesized that AMPKα2 KO muscle
displays impaired regulation of the PDH complex. PDK4
protein expression in quadriceps muscle was similar
between WT and AMPKα2 KO muscle at rest. Inter-
estingly, PDK4 protein expression was increased by 63%
with exercise in WT muscle only (Fig. 4A, P < 0.001)
compared to Rest 1. Six hours post-exercise, PDK4
protein expression was not statistically different from
Rest 2 in both genotypes, but tended (P = 0.06) towards
significance in WT. PDK4 protein was unaltered with
exercise and in recovery in AMPKα2 KO mice (Fig. 4A).
PDH-E1α Ser293 (Fig. 4B) and Ser300 phosphorylation
(Fig. 4C) in muscle increased (P < 0.05; P < 0.01,
respectively) with exercise independently of genotype with
a trend (P = 0.09) towards a larger increase in PDH-E1α
Ser293 phosphorylation in WT mice than in AMPKα2 KO
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
J Physiol 593.21 AMPK controls substrate metabolism during post-exercise recovery 4773
mice (Fig. 4B). Six hours post-exercise PDH-E1α Ser293
and Ser300 phosphorylation was at Rest 2 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 < 0.05) in WT compared to
AMPKα2 KO at all time points (Fig. 4D) and decreased
(P < 0.001) with exercise, independently of genotype
(Fig. 4D). Therefore, AMPKα2 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 Rest 2 levels in both WT
and AMPKα2 KO (Fig. 4D). Muscle acetyl-CoA content
(Fig. 4E) and the acetyl-CoA/free CoA ratio in muscle
(Table 2) were similar in WT and AMPKα2 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 < 0.01
and P < 0.001, respectively) with exercise independently of
genotype and were similar to Rest 2 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
WT and AMPKα2 KO at rest and increased (P = 0.05)
with exercise independently of genotype (Fig. 5A). Muscle
NAD+ content was similar in the two genotypes at rest
and increased (P < 0.001) 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 < 0.05) in p53 Lys379 acetylation
in quadriceps muscle (Fig. 5C) implying similar SIRT1
activation with exercise in WT and AMPKα2 KO muscle.
HDAC4 Ser632 phosphorylation in quadriceps was similar
in the two genotypes at rest and decreased (P < 0.05)
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-107, a proposed regulator of PDK4 trans-
lation, was measured. However, miR-107 level did not
change with exercise and was not different between
genotypes (Fig. 5E). RNU6B, snoRNA135, snoRNA202
and snoRNA234 mRNA levels were determined as
potential endogenous controls, but they all changed
with either exercise or genotype. Thus, miR-107 is not
shown as normalized to any endogenous control. Even
so, when miR-107 level was related to each of the
investigated controls, miR-107 still did not change with
exercise and was not different between genotypes, thus
25
Muscle glycogen
Free carnitine
M
u
s
c
le
 g
ly
c
o
g
e
n
 c
o
n
te
n
t 
(m
m
o
l/
k
g
 w
.w
.)
F
re
e
 c
a
rn
it
in
e
 (
m
m
o
l/
k
g
. 
d
.w
.)
F
re
e
 C
o
A
 (
n
m
o
l/
m
g
 w
.w
.)
M
a
lo
n
y
l-
C
o
A
 (
n
m
o
l/
m
g
 w
.w
.)
20
15
10
5
0
20
Free CoA
Malonyl-CoA
15
10
5
0
1.0
0.8
0.6
0.4
0.2
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
WT
A B
C D
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
RecoveryRest 2Rest 1 Exercise
RecoveryRest 2Rest 1 Exercise RecoveryRest 2Rest 1 Exercise
RecoveryRest 2Rest 1 Exercise
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 = 7–10. #P < 0.05, ###P < 0.001 main effect of exercise. ∗∗∗P < 0.001
main effect of genotype.w.w., wet weight; d.w., dry weight. From one litter, WT and AMPKα2 KO mice were
allocated into a basal, resting group (Rest 1) and an exercise group. From another litter, WT and AMPKα2 KO were
allocated into a basal, resting group (Rest 2) and a recovery group.
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
4774 A. M. Fritzen and others J Physiol 593.21
underlining no changes in miR-107 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. 1990; 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. 2009;
O’Neill et al. 2011; Jeppesen et al. 2013; Fentz et al. 2015).
Therefore, we chose to let mice lacking AMPKα2 and WT
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 O2 consumption
(V̇O2 ) was performed by WT 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 80% of energy
production) during prolonged and glycogen-depleting
exercise. However, immediately post-exercise AMPKα2
KO mice displayed a markedly suppressed FA utilization
compared to WT. 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
R1 R1 R2 R2Rec RecEx Ex
37 kDa
37 kDa
37 kDa
37 kDa
AMPKα2KO AMPKα2KOWT WT
pPDH Ser293
PDK4
pPDH Ser300
PDH-E1α
Coomassie
Rest 1 Rest 2Exercise Recovery
Rest 1 Rest 2Exercise Recovery
Rest 1 Rest 2Exercise Recovery
Rest 1 Rest 2Exercise Recovery
Rest 1 Rest 2Exercise Recovery
8
6
4
2
0
100
80
60
40
20
0
2.0
A
B
C
D
E
F
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
N
A
D
H
 (
n
m
o
l/
m
g
 d
.w
.)
A
c
e
ty
l-
C
o
A
 (
n
m
o
l/
m
g
 w
.w
.)
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
P
D
K
4
 p
ro
te
in
 (
A
U
)
p
P
D
H
 S
e
r
/P
D
H
-E
1
α 
(A
U
)
p
P
D
H
 S
e
r
/P
D
H
-E
1
α 
(A
U
)
PDH phosphorylation
PDH phosphorylation
PDK4 protein NADH
Acetyl-CoA
Figure 4. AMPKα2 KO mice have blunted exercise-induced PDK4 protein expression and lower NADH
content in muscle
PDK4 protein expression (A), PDH-E1α Ser293 phosphorylation/PDH-E1α protein (B), PDH-E1α Ser300
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 = 7–10. ##P < 0.01, ###P < 0.001 main effect of exercise (D) and significantly different from
rest within WT (A); ∗P < 0.05, ∗∗P < 0.01 main effect of genotype; ∗∗∗P < 0.001 significantly different from WT
within exercise. AU, arbitrary units. From one litter, WT and AMPKα2 KO mice were allocated into a basal, resting
group (Rest 1/R1) and an exercise group (Ex). From another litter, WT and AMPKα2 KO were allocated into a basal,
resting group (Rest 2/R2) and a recovery group (Rec).
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
J Physiol 593.21 AMPK controls substrate metabolism during post-exercise recovery 4775
Ta
b
le
2.
M
et
ab
o
lit
es
in
th
e
q
u
ad
ri
ce
p
s
m
u
sc
le
at
re
st
,a
ft
er
ex
er
ci
se
an
d
af
te
r
6
h
re
co
ve
ry
W
T
A
M
PK
α
2
K
O
W
T
A
M
PK
α
2
K
O
R
es
t
1
Ex
er
ci
se
R
es
t
1
Ex
er
ci
se
R
es
t
2
R
ec
o
ve
ry
R
es
t
2
R
ec
o
ve
ry
Su
cc
in
yl
-C
o
A
(m
m
o
l(
m
g
w
et
w
t)
−1
)
8.
07
±
0.
56
6.
56
±
0.
66
##
7.
54
±
0.
97
4.
89
±
0.
20
##
7.
24
±
0.
57
7.
05
±
0.
69
7.
64
±
0.
75
6.
03
±
0.
31
A
ce
ty
lC
o
A
/f
re
e
C
o
A
0.
28
±
0.
01
0.
29
±
0.
03
0.
31
±
0.
02
0.
28
±
0.
02
0.
33
±
0.
02
0.
32
±
0.
01
0.
30
±
0.
01
0.
33
±
0.
04
N
A
D
H
/N
A
D
+
2.
35
±
0.
23
0.
85
±
0.
14
##
#
1.
68
±
0.
23
0.
75
±
0.
14
##
#
2.
62
±
0.
31
2.
76
±
0.
40
3.
69
±
0.
73
3.
51
±
0.
34
D
at
a
ar
e
p
re
se
n
te
d
as
m
ea
n
s
±
SE
M
.n
=
7–
10
.#
#
P
<
0.
01
,#
##
P
<
0.
00
1
m
ai
n
ef
fe
ct
o
f
ex
er
ci
se
.F
ro
m
o
n
e
lit
te
r,
W
T
an
d
A
M
PK
α
2
K
O
m
ic
e
w
er
e
al
lo
ca
te
d
in
to
a
b
as
al
,r
es
ti
n
g
g
ro
u
p
(R
es
t
1)
an
d
an
ex
er
ci
se
g
ro
u
p
.F
ro
m
an
o
th
er
lit
te
r,
W
T
an
d
A
M
PK
α
2
K
O
w
er
e
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es
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.
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.
2012) indicating an important role of PDK4 in metabolic
regulation during exercise. In the same mice, a 50%
decreased liver but not muscle glycogen synthesis rate was
observed in the hours after exercise when pair-fed (Herbst
et al. 2014) 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 WT during
post-exercise recovery (Herbst et al. 2014), which in itself
indicates an importance of PDK4 during post-exercise
recovery. Moreover, the WT 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 WT mice, but inter-
estingly not in AMPKα2 deficient mice. This finding was
further supported by the observation of an increased
phosphorylation of PDH-E1α at Ser293 during exercise in
WT mice, but not AMPKα2 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 AMPKα2 deficient muscles generally have reduced
NADH content strengthening the point that AMPKα2 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. 2008; Li et al. 2009; White & Schenk, 2012).
The mechanism by which AMPK activation during
exercise regulates PDK4 protein content in skeletal muscle
could be in multiple steps from transcription to trans-
lation in the synthesis process of new protein as wellas stability of the protein. In the present study, PDK4
mRNA was shown to increase (P = 0.05) after a single
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
4776 A. M. Fritzen and others J Physiol 593.21
bout of exercise in both AMPKα2 KO and WT 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. 2005). Together these findings
clearly demonstrate that defective AMPKα2 does not
robustly affect exercise-induced gene expression of PDK4.
However, mRNA levels do not necessarily reflect trans-
criptional 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. 2003; Furuyama et al.
2003; Brunet et al. 2004; Gerhart-Hines et al. 2007;
Cantó et al. 2009; Mihaylova et al. 2011), were studied.
Exercise 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. 2011) 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 AMPKα2
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. 2008)
and mouse muscle deficient in AMPKγ3 performing
swimming exercise (Cantó et al. 2010) showing AMPK to
be necessary for activation of SIRT1. These discrepancies
are difficult to explain, but could be due to compensatory
up-regulation of AMPKα1 protein expression in AMPKα2
KO muscle (Jørgensen et al. 2005, 2007) or more likely a
difference in the response to different exercise regimes. The
robust finding of an increased PDK4 protein expression in
muscle of WT mice already after 2 h of exercise is striking.
Previous studies have shown that 40–90 min of treadmill
running in mice (Jørgensen et al. 2005; Cantó et al. 2009;
Kiilerich et al. 2010) and 60–150 min of exercise in humans
(Pilegaard et al. 2000, 2002, 2005) 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 WT mice, despite similar
PDK4 mRNA levels between WT and AMPKα2
KO muscle, point towards a role of AMPK in
post-transcriptional regulation of PDK4 protein. Recently,
4
A B C
D E F
P=0.05
3
2
1
0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2.0
1.5
1.0
0.5
0.0
80
60
40
20
0
p
H
D
A
C
4
 S
e
r6
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2
/H
D
A
C
4
 (
A
U
)
P
D
K
4
 m
R
N
A
/s
s
D
N
A
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D
+
c
o
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te
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t 
(n
m
o
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m
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 d
.w
.)
A
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ty
la
ti
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 p
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3
 L
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 p
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 (
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 (
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Rest Rest Exercise
Rest Exercise Rest Exercise
HDAC4 phosphorylation
PDK4 mRNA NAD+
miR-107
p53 acetylation
Rest Exercise
Exercise
WT
AMPKα KO
WT
AMPKα KO
WT
AMPKα KO
Re ReEx Ex
50 kDa
50 kDa
100 kDa
100 kDa
AMPKα2KOWT
p53
a-p53
pHDAC4
HDAC4
WT
AMPKα KO
WT
AMPKα KO
Figure 5. Similar exercise-induced increase in PDK4 mRNA levels, p53 acetylation and decreased HDAC4
phosphorylation in AMPKα2 KO and WT muscle
PDK4 mRNA levels (A), NAD+ content (B), p53 Lys379 acetylation/p53 protein (C), HDAC4 Ser632
phosphorylation/HDAC4 protein (D), and miR-107 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 = 7–10.
#P < 0.05, ##P < 0.01, ###P < 0.001 main effect of exercise. AU, arbitrary units; Ex, exercise; d.w., dry weight;
Re, rest.
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society
J Physiol 593.21 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. 2013) suggesting a role for AMPK in translational
regulation through miR-mediated regulation. miR-107
was predicted to regulate PDK4 translation (Wilfred
et al. 2007) and suggested to be important in the
regulation of PDK4 protein content after exercise in mouse
muscle (Safdar et al. 2009). However, the observation
that miR-107 expression was independent of genotype
and not affected by exercise suggests that AMPKα2
does not regulate the protein abundance of PDK4 via a
miR-107-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 WT and AMPKα2 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. 2001; Roepstorff et al. 2005; Jeppesen et al.
2013), the form required for mitochondrial FA trans-
membrane 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
AMPK
γ
NADH PDK4 pyruvate
acetyl-CoA
FA OXGLU OX
PDH
Thr172
AM
P
α2
β
Figure 6. Scheme of proposed AMPK-mediated regulation of
fuel selection during post-exercise recovery
Exercise-induced increase in AMPKα2 activity in skeletal muscle
increases pyruvate dehydrogenase 4 (PDK4) protein content, and
AMPKα2 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 AMPKα2 KO mice. This is in line with earlier
observations showing lower muscle glycogen levels during
the post-exercise recovery period in muscle lacking
functional AMPKα2 (Mu et al. 2003; Jørgensen et al.
2005) or AMPKγ3 (Barnes et al. 2004), 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 AMPKα2 KO mice is similar to WT (Viollet
et al. 2003). Moreover, in the present study, WT mice
utilized a greater amount of energy and muscle glycogen
during the exercise bout at the same relative intensity as
AMPKα2 KO mice. If anything this would be expected
to lead to a higher intake of carbohydrate-rich chow
food, resulting in a higher RER in WT post-exercise.
But the opposite was observed. Thus, taken together, it
seems unlikely that potential differencesin food intake
post-exercise can explain the observed findings in the pre-
sent 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 AMPKα2 plays an
important, but indirect role, in increasing FA oxidation
in muscle during post-exercise recovery. This seems to
occur through an AMPKα2–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.
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Additional information
Competing interests
The authors declare there are no conflicts of interest.
Author contributions
A.M.F., J.J., M.L.B.C. and B.K. designed and conceived the study.
A.M.F., J.J., M.L.B.C., A.-M.L., R.B. and H.P. collected and
analysed data. The experiments were carried out at the University
of Copenhagen, Denmark, in the Molecular Physiology Group,
Department of Nutrition, Exercise and Sports. A.M.F. and B.K.
wrote the manuscript with all co-authors revising it critically
for important intellectual content. All authors have approved
the final version of the manuscript and agree to be accountable
for all aspects of the work. All persons designated as authors
qualify for authorship, and all those who qualify for authorship
are listed.
Funding
The study was supported by funding from the Novo Nordisk
Foundation (R168-A14319 to B.K.), the Lundbeck Foundation
(R17-A1760 to B.K.) and The Danish Council for Independent
Research – Natural Sciences (1323-00317A to H.P.). The PhD
Scholarship of Anne-Marie Lundsgaard was funded by The
Danish Diabetes Academy which is supported by the Novo
Nordisk Foundation.
Acknowledgements
The authors are grateful for the skilled technical assistance
of I.B. Nielsen, Molecular Physiology Group, Department
of Exercise and Sport Sciences, University of Copenhagen.
The authors thank Professor B. Viollet (Department of End-
ocrinology, Metabolism and Cancer, Institut Cochin, Université
Paris Decartes, Paris, France) for providing the AMPKα1
and AMPKα2 KO founder mice. We also thank D. Grahame
Hardie (Dundee University, UK) for the kind donation of the
anti-AMPKα1, anti-AMPKα2, anti-PDH-1Eα, anti-pPDH-1Eα
Ser293, anti-pPDH-1Eα Ser300 and anti-PDK4 antibodies for this
study.
C© 2015 The Authors. The Journal of Physiology C© 2015 The Physiological Society