D.

Grahame Hardie and Kei Sakamoto

Physiology 21:48-60, 2006. doi:10.1152/physiol.00044.2005

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REVIEWS
AMPK: A Key Sensor of Fuel and Energy
Status in Skeletal Muscle
Contraction induces marked metabolic changes in muscle, and the AMP-activated
protein kinase (AMPK) is a good candidate to explain these effects. Recent work
using a muscle-specific knockout of the upstream kinase, LKB1, has confirmed that
the LKB1AMPK cascade is the signaling pathway responsible for many of these
effects.
It has been known for many years that exercise and
contraction induces marked changes in the metabo-
lism of skeletal muscle, including increases in glycogen
breakdown, glycolysis, glucose uptake, and fatty acid
oxidation, together with many changes in gene expres-
sion. The mechanism by which contraction triggers
glycogen breakdown was established by Krebs and
Fischer 50 years ago, when they reported that glycogen
phosphorylase was activated by a Ca2+-activated pro-
tein kinase [now termed phosphorylase kinase (70)].
However, the signaling pathways responsible for most
of the other metabolic changes induced by exercise or
contraction, including the increased glucose uptake
and fatty acid oxidation, remained enigmatic. The first
clue that the AMP-activated protein kinase (AMPK)
might play an important role came from findings in
Winder’s laboratory (116) that malonyl-CoA levels
decreased during exercise in rat muscle. Since mal-
onyl-CoA was known to be an inhibitor of fatty acid
uptake into mitochondria [via inhibition of
carnitine:palmitoyl-CoA acyl transferase (CPT1) on the
outer surface of the inner mitochondrial membrane, a
mechanism established by the late Dennis McGarry
(78) and shown in FIGURE 1], this potentially
explained the increase in fatty acid oxidation induced
by exercise. However, what was the cause of the
decrease in malonyl-CoA? This metabolite is produced
by the enzyme acetyl-CoA carboxylase, which occurs
as two isoforms, i.e., ACC1 and ACC2, thought to pro-
duce malonyl-CoA for fatty acid synthesis and for regu-
lation of fatty acid oxidation, respectively, with ACC2
being the form expressed in muscle (2). ACC1 was
already known to be phosphorylated and inactivated
by AMPK in response to ATP depletion in liver cells (25,
27), suggesting that ACC2 might also be inactivated by
AMPK during muscle contraction. Activation of AMPK
in skeletal muscle, with simultaneous inactivation of
ACC2 and decreased malonyl-CoA, was indeed
demonstrated in response to both exercise in vivo (117)
and contraction induced by electrical stimulation of
the sciatic nerve in situ (55, 114).
A method to demonstrate a more direct, causal rela-
tionship came with the development of 5-aminoimi-
dazole-4-carboxamide riboside (AICAR) as a pharma-
cological agent to activate AMPK in intact cells and tis-
48 1548-9213/06 8.00 ©2006 Int. Union Physiol. Sci./Am. Physiol. Soc.
PHYSIOLOGY 21: 48–60, 2006; 10.1152/physiol.00044.2005
D. Grahame Hardie1
and Kei Sakamoto2
1Division of Molecular Physiology and
2MRC Protein Phosphorylation Unit,
University of Dundee, Dundee, Scotland
d.g.hardie@dundee.ac.uk
sues (23). Perfusion of rat hindlimb muscle with
AICAR caused AMPK activation, and, as expected, this
correlated with inactivation of acetyl-CoA carboxy-
lase, decreased malonyl-CoA, and increased fatty acid
oxidation (79) (FIGURE 1). In the same study, glucose
Downloaded from physiologyonline.physiology.org on October 27, 2010
uptake was also measured and found to increase (79),
an effect that was subsequently shown to be due to
increased transport across the plasma membrane (47)
and to involve translocation of GLUT4 to the plasma
membrane (72). These results demonstrated that acti-
vation of AMPK in muscle was sufficient for increased
glucose uptake, but not that it was necessary for the
increased glucose uptake observed during contrac-
tion. Before discussing the recent evidence that
strongly supports that idea, we need to introduce the
AMPK system itself in more detail.
AMPK: Early Studies
AMPK has been recently reviewed elsewhere (35, 38,
40, 65), and in this article we focus on studies particu-
larly relevant to skeletal muscle. The kinase was first
defined (20, 37) as a protein kinase that phosphorylat-
ed and inactivated both 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase and ACC1 (regulatory enzymes
of cholesterol and fatty acid synthesis, respectively),
although it was subsequently shown to inactivate
ACC2 as well (119). As its name suggests, AMPK is
allosterically activated by 5’-AMP (20), an effect antag-
onized by high concentrations of ATP (23). Because of
the reaction catalyzed by adenylate kinase (2ADP

ATP + AMP), the AMP:ATP ratio varies approximately
as the square of the ADP:ATP ratio (38), making the
former ratio (essentially the parameter to which AMPK
responds) a sensitive indicator of reduced cellular
energy status. Consequently, any cellular or metabolic
stress that either inhibits ATP synthesis [e.g., heat
shock (22), hypoxia (77), ischemia (71), or glucose
deprivation (102)] or that accelerates ATP consump-
tion [e.g., contraction of skeletal muscle (55, 114, 117)]
causes AMPK activation. It had been known for many
years that muscle glycogen phosphorylase and phos-
phofructokinase (the key enzymes regulating glycogen
breakdown and glycolysis, respectively) can also be
activated allosterically by a rise in the AMP:ATP ratio

(21, 91). The idea that this ratio was a key indicator of
cellular energy status was therefore not new. However,
the concept that a single protein kinase like AMPK
could sense the same parameters and transmit the
information to potentially hundreds of downstream
targets was an important new development.
AMPK: Subunit Structure and
Regulation by Upstream Kinases
AMPK is now known to exist as heterotrimeric com-
plexes containing a catalytic ␣-subunit and regulatory
␤- and ␥-subunits. Each subunit is encoded by multi-
ple genes (␣1, ␣2, ␤1, ␤2, ␥1, ␥2 and ␥3), and some of
the mRNAs are also subject to alternative splicing, giv-
ing rise to a large variety of possible heterotrimeric
combinations. The complex that predominates in
most cell types appears to be the ␣1␤1␥1 combination,
but skeletal muscle is unique in that it may be the only
tissue that expresses all subunit isoforms, including ␥3
(76). Different isoform combinations are stimulated to
various extents by AMP and also appear to differ in
their subcellular localization (101).
Although AMPK received its name because of the
allosteric activation by AMP, this effect is rather small
(5-fold or less) and insignificant compared with the
effect of phosphorylation by upstream kinases. These
modify a specific threonine residue (Thr172) within the
activation loop of the kinase domain on the ␣-subunit,
whose phosphorylation is essential for kinase activity
(43, 105, 108). Significantly, the level of phosphoryla-
tion at this site is also exquisitely regulated by AMP,
both due to stimulation of phosphorylation by the
upstream kinase (46) and to inhibition of dephospho-
rylation by protein phosphatases (26), both effects
being antagonized by high concentrations of ATP.
Mitochondrion
Outer
membrane
Matrix Inner
membrane
FIGURE 1. Activation of muscle fatty acid oxi-
dation by AMPK
AMP-activated protein kinase (AMPK), whether acti-
vated by exercise or by 5-aminoimidazole-4-carbox-
amide riboside (AICAR), phosphorylates and inacti-
vates the ACC2 isoform of acetyl-CoA carboxylase
(119) [which is associated with the mitochondrial
membrane (1)], thus lowering malonyl-CoA. This
relieves the inhibition of CPT1 (78), allowing fatty acids to enter mitochondria as carnitine esters (FA-carnitine). They are converted by
carnitine:palmitoyl-CoA acyl transferase 2 (CPT2) back to fatty acyl-CoA (FA-CoA) esters in the mitochondrial matrix, where they are oxidized to gen-
erate ATP.
REVIEWS
These effects are entirely substrate mediated, i.e., they
are due to binding of the nucleotides to the substrate
(AMPK) and not to the upstream kinase or the protein
phosphatase. Working together, the three effects of
AMP mean that the system is activated by increases in
the cellular AMP:ATP ratio in a highly sensitive man-
ner (39).
Because a rise in AMP triggers phosphorylation and
activation of AMPK, any metabolic poison that
inhibits mitochondrial ATP synthesis (e.g., Refs. 22, 44,
120) will activate AMPK by increasing the cellular
AMP:ATP ratio. Intriguingly, the biguanide drug met-
formin, and thiazolidinedione drugs like rosiglitazone
(two of the currently most widely prescribed drugs for
type 2 diabetes) have both been found to activate
AMPK (33, 131). Whereas thiazolidinediones have at
least one other target [the peroxisome proliferator-
activated receptor-␥ (PPAR-␥)], it seems likely that
Downloaded from physiologyonline.physiology.org on October 27, 2010
AMPK activation explains most, if not all, of the thera-
peutic actions of metformin. Metformin, the related
biguanide phenformin, and the thiazolidinediones are
all inhibitors of complex I of the mitochondrial respi-
ratory chain (17, 28, 89). It seems likely that these
drugs activate AMPK indirectly by increasing the cel-
lular AMP:ATP ratio, which has indeed been demon-
strated for rosiglitazone (33) and phenformin (45),
although not for metformin (33, 44).
Functions of the ␥-Subunits and
Mutations Causing Human Disorders
AMPK is a highly conserved protein kinase, and genes
encoding the ␣-, ␤-, and ␥-subunits can be found in all
eukaryotic genomes, including primitive protozoa,
fungi, and plants, as well as mammals (40). The ␣-sub-
units are unstable on their own and the ␤- and ␥-sub-
Matrix Inner membrane Intramembrane space/
ADP cytosol
ATP
FA-CoA Carnitine FA-CoA
O2 CPT-2 CPT-1 Malonyl-CoA
carboxylase-2
FA-carnitine
Acetyl-CoA
CO2 CoA CoA
AMPK
Acetyl-
CoA
Exercise AICAR
PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org 49
REVIEWS
units are essential for activity. A highly conserved fea-
ture of all ␥-subunits is the two repeats of a sequence
motif now known as a Bateman domain, each of which
is itself composed of two repeated CBS motifs (67).
Single copies of this domain are found in a small num-
ber of proteins other than the AMPK ␥-subunits, and it
is becoming clear that their function is to bind adeno-
sine-containing ligands such as AMP, ATP, or S-adeno-
syl methionine (104). Bacterially expressed constructs
containing the NH2-terminal or COOH-terminal
Bateman domains from ␥2 each bind one molecule of
AMP or ATP in a mutually exclusive manner, whereas
a construct containing both domains binds two mole-
cules of AMP or ATP with strong positive cooperativity
(104).
Are the Bateman domains responsible for the regu-
latory effects of AMP on AMPK phosphorylation and
activity? The best evidence for this comes from studies
of mutations in the human ␥2 gene that cause heart
disease. At least six different point mutations occur-
ring in the two Bateman domains cause a form of
hereditary Wolff-Parkinson-White (WPW) syndrome
(104), a cardiac arrhythmia characterized by ventricu-
lar preexcitation, in which the normal delay between
the contraction of the atria and ventricles is reduced.
Subjects experience frequent arrhythmic episodes and
can die from heart failure at an early age. Studies using
a transgenic mouse model of one of the mutants sug-
gest that the arrhythmias may be secondary to an ele-
vated glycogen storage in the cardiac muscle (7), and it
is interesting to note that one of the classical glycogen
storage disorders (Pompe’s disease, due to lysosomal
␣14 glucosidase deficiency) can present with very
similar clinical features (32). More recently, three
unrelated human subjects were identified with a novel
“Our current hypothesis is that this high AMPK
activity leads to a higher basal glucose uptake
into the myocytes, with excessive
glycogen storage being a consequence of this.”
␥2 mutation that caused a much more severe form of
heart disease (18). This mutation (R531Q) occurs at the
same residue that is mutated in one of the inherited
disorders (R531G). However, this mutation is not inher-
ited because the subjects all died within days of birth
and, in one case where both parental DNAs were avail-
able, they were normal. At autopsy the hearts of these
infants were found to be grossly enlarged, with a very
high glycogen content that was clearly disrupting the
normal organization of the myofibrils, supporting the
idea that the primary effect of ␥2 mutations is to cause
a glycogen-storage disorder. Interestingly, a mutation
50 PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org
in the pig ␥3 gene causes an amino acid replacement
(R200Q) that is exactly equivalent to one of the muta-
tions (R302Q) in human ␥2 causing WPW syndrome
(80). The ␥3 gene appears to be expressed exclusively
in skeletal muscle, and the R200Q mutation gives rise to
elevated glycogen storage in that tissue. Unlike the
harmful effects of elevated glycogen in cardiac muscle,
in skeletal muscle it appears to be well tolerated and
even increases resistance to fatigue (12).
What is the effect of these mutations on the binding
of AMP to AMPK and its regulation by the nucleotide?
Consistent with the idea that the Bateman domains
are the regulatory AMP-binding sites, all of the muta-
tions affect both binding of AMP to the isolated
Bateman domains and activation of the ␣␤␥-complex-
es by AMP (24, 104). Moreover, the order of severity of
the mutations is the same for both effects (18, 104).
This provides strong evidence that the Bateman
Downloaded from physiologyonline.physiology.org on October 27, 2010
domains are responsible for the activating effects of
AMP. It is also noticeable that the mutation that has
the greatest effect on AMP and ATP binding (R531Q)
also causes the most severe clinical defect (18). Four of
the mutations (R302Q, H383R, R531G/R531Q) affect basic
residues, and modeling of the Bateman domains sug-
gests that their positively charged side chains bind the
negatively charged phosphate group of AMP (104).
The fact that mutations in both Bateman domains can
affect activation by AMP suggests that both sites need
to be occupied before AMPK is activated, and since
binding to the two sites is highly cooperative, this
would be another mechanism to increase the sensitiv-
ity of the AMPK system. The Bateman domains also
bind ATP (albeit at lower affinity than AMP), with
binding of AMP and ATP being mutually exclusive.
This is consistent with the previous findings that high
concentrations of ATP inhibit all three activating
effects of AMP on the AMPK system (23, 26, 43).
One puzzling feature of the ␥-subunit mutations is
that, although lack of activation by AMP represents a
loss of function, the mutations are dominant in their
effect. However, the mutations also reduce binding of
the inhibitor, ATP, as well as the activator, AMP, so the
overall effect will depend on the relative concentra-
tions of the two nucleotides in vivo (104). In fact, coex-
pression of the R531G and R531Q mutants with ␣1 and
␥1 in human embryonic kidney 293 cells shows that,
although the mutant complexes are no longer activat-
ed by cellular stresses, their basal level of phosphory-
lation and activity is higher than that of the wild type,
possibly because inhibition of phosphorylation by
basal levels of ATP is relieved (18). Our current
hypothesis is that this high AMPK activity leads to a
higher basal glucose uptake into the myocytes, with
excessive glycogen storage being a consequence of
this.
The ␥-subunits of AMPK are not the only muscle
proteins containing Bateman domains. The nine
members of the human ClC chloride channel family

all contain a single Bateman domain at the COOH ter-
minus, which is cytoplasmic. We have recently shown
that the Bateman domain of ClC2 (an isoform
expressed in neurons and other cells) binds ATP and
that a mutation in ClC2 associated with idiopathic
generalized epilepsy, as well as a different mutation
equivalent to one found in the muscle isoform, ClC1,
drastically reduce this binding (104). The mutation in
ClC1 causes congenital myotonia, a form of muscle
stiffness characterized by a delayed relaxation after
contraction. Very recently, Bennetts et al. (13) have
shown that ATP binding reduces the open probability
of ClC1 at resting membrane potentials. Since ClC1 is
found at the plasmalemma and in T tubules, opening
of its anion channel would stabilize the resting mem-
brane potential and repolarize the plasmalemma fol-
lowing excitation by acetylcholine. Bennetts et al. (13)
propose that the opening of the ClC1 channel would
represent a mechanism to limit muscle contraction as
ATP levels fall and may therefore be a factor in muscle
fatigue. This feedback mechanism would be defective
in the subjects with congenital myotonia. Although
this effect is not mediated by AMPK, it involves an
“energy-sensing” domain very similar to those found
on the ␥-subunits of AMPK.
Functions of the ␤-Subunits and
Regulation of AMPK by Glycogen
As well as a COOH-terminal domain that is required
for binding to the ␣- and ␥-subunits (53, 60), all ␤-sub-
units contain a conserved central domain that has
been shown to bind glycogen (53, 93). Although this
glycogen-binding domain (GBD) is conserved across
all eukaryotic species and certainly causes interac-
tions between AMPK and glycogen particles in intact
cells, its physiological function remains unclear.
Glycogen synthase appears to be a physiological target
for AMPK in skeletal muscle (see below), and one pos-
sibility is that the GBD colocalizes AMPK with this key
substrate. A second intriguing possibility, not neces-
sarily mutually exclusive with the first, is that the
AMPK system can act as a sensor not just of the short-
term energy status of the cell in the form of ATP but
also of the medium-term reserves of energy in the
form of glycogen. There is some evidence in favor of
this idea, in that a high level of muscle glycogen
represses the activation of AMPK by AICAR in per-
fused rat muscle (122) and by exercise in human mus-
cle (121).
Identification of Upstream Kinases
A major breakthrough in the study of the regulation of
AMPK in the past couple of years has been the first
definitive identification of upstream kinases that
phosphorylate Thr172 on the ␣-subunit and thus acti-
vate the kinase. Initial advances came when various
REVIEWS
genome-wide screening approaches identified three
kinases, i.e., Elm1, Pak1 and Tos3, that act upstream of
the yeast ortholog of AMPK, the SNF1 complex (52, 85,
109). The nearest relatives of these yeast kinases in
mammals were LKB1 and the ␤-isoform of calmod-
ulin-dependent protein kinase (CaMKK-␤).
Subsequent work has shown that LKB1 and CaMKK-␤
(and perhaps also CaMKK-␣) can indeed be physio-
logical activators of AMPK under different circum-
stances in vivo.
Intriguingly, LKB1 was originally identified as the
gene mutated in the rare autosomal dominant human
genetic disorder, Peutz-Jeghers syndrome (PJS) (48,
59). PJS subjects develop numerous benign tumors
(classed as hamartomas) in the gastrointestinal tract
and have a 20-fold-increased risk of developing malig-
nant tumors at other sites (34), whereas mutations in
the LKB1 gene are also seen in some sporadic cancers,
Downloaded from physiologyonline.physiology.org on October 27, 2010
especially adenocarcinoma of the lung (103). LKB1 is
thus a classical tumor suppressor. The functional form
of LKB1 is a complex with two accessory subunits,
Ste20-related adaptor protein (STRAD) (11) and
Mouse protein-25 (MO25) (15), both of which exist as
two isoforms (␣ and ␤) encoded by distinct genes. One
of us (D. G. Hardie) had partially purified an upstream
kinase for AMPK from rat liver (43), and following the
new findings in the yeast system our laboratories were
able to show that this was a complex between LKB1,
STRAD-␣, and MO25-␣ (42). LKB1 also exists as a
complex with STRAD-␣ and MO25-␣ in skeletal mus-
cle (K. Sakamoto, J. Boudeau, and D. Alessi, unpub-
lished observation). Experiments with cultured cells
in which the expression of LKB1 was manipulated in
various ways proved that it was both necessary and
sufficient for the activation of AMPK by AICAR and
phenformin (42, 107, 124). Experiments revealing the
important role of LKB1 in muscle will be discussed in
a later section.
Does activation of AMPK explain the tumor-sup-
pressor effects of LKB1? This seems possible, because
activation of AMPK has been reported to inhibit the
target of rapamycin (TOR) pathway, thus potentially
inhibiting cell growth and hypertrophy (see below)
while it also blocks the G1
S phase transition in the
cell cycle (61). However, LKB1 acts upstream of a fam-
ily of at least 12 AMPK-related kinases (58, 73), and
some of these could also be involved in the tumor-sup-
pressor effects of LKB1. Another interesting question
is whether PJS patients display any metabolic disorder.
To date, about 150 mutations in LKB1 have been iden-
tified in PJS patients and sporadic cancers (4, 16). The
majority of these result in substantial truncations of
the catalytic domain and would be expected to impair
LKB1 catalytic activity. It would be interesting to find
out if the activity of AMPK in tissues, including muscle
and liver, is reduced in PJS patients, and if so whether
this would affect their metabolic responses during
exercise.
PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org 51
REVIEWS
Recently, three groups simultaneously reported evi-
dence that the CaMKKs can act upstream of AMPK, at
least in some cell types (45, 54, 123). In cells lacking
expression of LKB1, phosphorylation of AMPK at
Thr172 and consequent activation did not occur in
response to AICAR or phenformin but still occurred in
response to Ca2+ ionophores. Experiments using phar-
macological inhibitors and short interfering RNAs
directed against the CaMKKs suggest that the latter,
and especially CaMKK-␤ (45, 123), are responsible for
activation of AMPK via phosphorylation of Thr172
under these circumstances. The failure of AICAR or
phenformin to activate AMPK in cells lacking LKB1,
but expressing CaMKKs, shows that LKB1 is essential
for activation of AMPK mediated by a rise in the
AMP:ATP ratio. It also suggests that a rise in AMP is
not sufficient to stimulate phosphorylation of AMPK
by the CaMKKs, which are activated instead by elevat-
ed Ca2+ concentrations.
What is the physiological relevance of these obser-
vations? The CaMKKs have a rather restricted tissue
distribution and are mainly expressed in cells of neu-
ral origin (5). In rat brain slices, depolarization of neu-
rons induced by increased medium K+ concentration
(known to cause an influx of Ca2+ by voltage-gated
channels) caused activation of AMPK that was blocked
by a CaMKK inhibitor and was not associated with
changes in the cellular AMP:ATP ratio. By contrast,
activation of AMPK by phenformin in the same system
was accompanied by increases in AMP:ATP and was
unaffected by the CaMKK inhibitor (45). The CaMKKs
are not expressed significantly in skeletal muscle, and
experiments with muscle-specific knockouts of LKB1
(see below) suggest that the CaMKK
AMPK pathway
cannot be a major player in mediating the effects of
muscle contraction, despite the fact that increases in
cytosolic Ca2+ obviously occur. In cells where the
CaMKKs are significantly expressed, such as neurons,
activation of AMPK may be a mechanism for antici-
pating the increased demand for ATP that always
accompanies rises in cytosolic Ca2+, due to the fact
that the Ca2+ must be pumped out of the cytoplasm to
restore basal levels.
Activation of AMPK in Muscle
Since the original observations that AMPK was activat-
ed in muscle during exercise (117) and electrically
stimulated contraction (55, 114), it has generally been
assumed that the activation is caused by an increase in
the cellular AMP:ATP ratio caused by increased ATP
consumption. Increases in this ratio have indeed been
observed during in situ electrical stimulation of mouse
muscle (99), although not after exercise in rat muscle
in vivo (117). However, any changes in the latter case
may have been lost during the time it took to remove
the muscle for analysis. Unfortunately, levels of mus-
cle AMP are too low to be measured noninvasively by
52 PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org
31
P nuclear magnetic resonance.
Even if an increase in AMP:ATP ratio is the primary
signal for AMPK activation during exercise, this does
not rule out the possibility of additional mechanisms.
The cytokine interleukin-6 (IL-6) is released from
muscle during exercise (29), and it has recently been
reported that it activates AMPK in isolated rat muscles
(66). Moreover, both basal and exercise-stimulated
AMPK activity were reduced in the gastrocnemius
muscle of IL-6 knockout mice, although the degree of
stimulation of AMPK by exercise was similar (66).
These results suggest that IL-6 may amplify the effects
of exercise on AMPK via an autocrine mechanism.
Other important recent results have shown that
AMPK is activated in muscle by the adipokines leptin
(82) and adiponectin (112, 128) and that this may be
responsible for the increase in fatty acid oxidation, and
hence (at least in part) the increase in energy expendi-
Downloaded from physiologyonline.physiology.org on October 27, 2010
ture, induced by these cytokines. Coupled with find-
ings that leptin regulates AMPK in the hypothalamus
and that hypothalamic AMPK regulates food intake (6,
81), these results show that AMPK is a key player in
regulation of energy balance not only at the cellular
level, but also at the whole body level.
Downstream Targets of AMPK
This topic has been discussed in several recent reviews
(35, 40, 65), and in this article we will focus on a small
number of recently identified targets that are particu-
larly relevant to skeletal muscle. Metabolic changes
induced by AMPK in muscle (summarized in FIGURE
2) are both acute changes due to direct phosphoryla-
tion of metabolic enzymes and chronic changes due to
effects on gene expression. In general, the metabolic
changes induced by AMPK are similar to those pro-
duced by endurance exercise (e.g., distance running),
such as increased uptake and oxidation of plasma glu-
cose and fatty acids and increased expression of the
glucose transporter GLUT4 and hexokinase (HKII)
(50). By contrast, the changes that occur in response to
resistance exercise (e.g., weightlifting), such as
increased glycogen breakdown and glycolysis, are
caused by regulation of phosphorylase and phospho-
fructokinase by established mechanisms that do not
require AMPK.
An important question, which has not been com-
pletely answered, is whether AMPK is preferentially
activated by endurance rather than resistance exer-
cise. Most of the protocols that have been used to
study regulation of AMPK in humans have involved
endurance exercise, and, perhaps surprisingly, no
direct comparisons of endurance vs. resistance exer-
cise have been performed. However, studies of rat
muscle using electrical stimulation favor the idea that
activation of AMPK occurs primarily in response to
endurance exercise. Firstly, AMPK activation by con-
tinuous low-frequency stimulation of rat gastrocne-
 REVIEWS

Glucose Fatty acids

Blood

Muscle GLUT 4 FAT/CD36

Glycogen
Glycogen
synthase ? ?
UDPG G1P G6P ADP
Gene ATP
expression Pyruvate Pyruvate
FA-CoA FA-CoA
CO2
Malonyl-CoA Acetyl-CoA
Acetyl-CoA
carboxylase
PGC-1␣
ATP ADP AMP AMPK

Downloaded from physiologyonline.physiology.org on October 27, 2010

Adenylate
? Cr PCr kinase
LKB1
TOR
ATP ADP

Protein synthesis
Muscle hypertrophy
Muscle contraction

FIGURE 2. Metabolic changes known to be induced by AMPK in muscle, including stimulation of glu-
cose and fatty acid uptake, fatty acid oxidation, and mitochondrial biogenesis, and inhibition of
glycogen synthesis and, via inhibition of TOR, hypertrophy
Question marks indicate that the direct target for AMPK responsible for the observed downstream effect is not
known. The effect on fatty acid uptake has to date only been observed in cardiac muscle. The mechanisms of inhibi-
tion of fatty acid oxidation and the target of rapamycin (TOR) by AMPK is shown in more detail in FIGURES 1 AND 3.

mius/plantaris in situ is rather slow, occurring in min-
utes rather than seconds (55). Secondly, a recent study
of ex vivo stimulation of rat extensor digitorum longus
and soleus muscles using protocols designed to simu-
late resistance or endurance exercise showed that
AMPK was not activated by the former (9). The “resist-
ance exercise” protocol involved short bursts of high-
frequency stimulation followed by rest periods, and it
may be that any tendency for AMPK to become acti-
vated during the bursts of stimulation was reversed
during the rest periods as ATP levels recovered. It
would be of interest to confirm these findings in
human muscle using different exercise protocols.
Taking everything together, AMPK appears not to be
required for anaerobic metabolism of endogenous
glycogen but is required instead for the switch to aero-
bic oxidation of blood-borne fuels. There are obvious
analogies between this and the role of the yeast
ortholog of AMPK, the SNF1 complex. Genetic analy-
sis shows that the SNF1 complex is required for the
switch from anaerobic (fermentative) metabolism of
glucose to the oxidative metabolism of glucose and
other fuels (36).
One consequence of resistance exercise training that
is not seen in response to endurance exercise is hyper-
trophy, i.e., increased muscle bulk due to increases in
protein synthesis and muscle fiber volume. Relevant to
this, a recently identified target for AMPK is the TOR
protein kinase pathway, which is activated by insulin,
growth factors, and amino acids and stimulates protein
synthesis, and hence cell growth and hypertrophy.
Using the phosphorylation of ribosomal protein S6
kinase as a marker for TOR activation, AMPK has been
found to inhibit its activation in several cell types
including skeletal muscle (14, 68, 69), probably via
phosphorylation of TSC2 (tuberin) (57). The latter
forms a complex with TSC1 (hamartin) and contains a
GTPase activator protein (GAP) domain that promotes
conversion of the small G protein Rheb to its GDP-
bound form, which no longer activates TOR (110)
(FIGURE 3). Ribosomal protein S6 kinase is activated
by an in vivo electrical stimulation protocol designed to
simulate resistance exercise and produce hypertrophy
in rat muscle (10), while studies using the TOR inhibitor
rapamycin (95) and mice in which S6 kinase is knocked
out (87) suggest that activation of TOR and S6 kinase are
both required for muscle hypertrophy. The ability of
AMPK to inhibit TOR thus provides a potential explana-
tion for the lack of muscle hypertrophy induced by
endurance exercise training. Muscles of mice with

PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org 53

REVIEWS
knockouts of the ␣1 (63, 64) or ␣2 (115) subunits of
AMPK or of LKB1 (99) do not display obvious hypertro-
phy, although their responses to resistance exercise or
growth factor treatment have not been studied. It is also
interesting that loss-of-function mutations in LKB1
(which acts upstream of AMPK) and of TSC1 or TSC2
(which act downstream of AMPK) both lead to a high
incidence of benign tumors classed as hamartomas in
humans; in the case of TSC1 and TSC2 the condition is
known as tuberous sclerosis complex (90).
Hamartomas are benign tumors that grow abnormally
but retain their differentiated status, indicating that
their defect is in regulation of cell growth.
A single bout of endurance exercise is capable of
increasing the insulin sensitivity of muscle, as first
demonstrated in 1982 (94). This effect of exercise can
be mimicked by activation of AMPK using AICA ribo-
side both in vivo (56) and in vitro (30). Although the
mechanism for this effect is not known, one intriguing
possibility is that it involves the ability of AMPK to
inhibit the TOR pathway. Recent studies in which the
activity of the TOR  ribosomal protein S6 kinase I
pathway was ablated by various approaches suggest
that that pathway downregulates the insulin signaling
pathway via phosphorylation of the insulin receptor
substrate-1 (IRS1). This phosphorylation downregu-
lates IRS1 function by reducing its synthesis, by
increasing its degradation, and/or by reducing its abil-
ity to activate phosphatidylinositol-3-kinase (41, 106,
113). AMPK activation has the potential to reverse this
feedback regulation on insulin signaling via its ability
to inhibit the TOR pathway.
One other consequence of endurance exercise train-
ing is an increase in oxidative capacity via increased
mitochondrial biogenesis, a phenomenon first
observed by Holloszy in 1967 (49). It is important to
AMP AMPK TSC2:TSC1
Rheb.GTP
LKB1 Rheb.GDP
TOR
S6K1 4EBP1
Protein synthesis
Muscle hypertrophy
FIGURE 3. Proposed mechanism of inhibition of
TOR, and hence protein synthesis and muscle
hypertrophy, by AMPK
AMPK phosphorylates TSC2 (hamartin) at two sites (57).
This is proposed to stimulate its GTPase activator pro-
tein (GAP) activity against the small G protein, Rheb,
converting the latter to its GDP-bound form that no
longer activates TOR. Two proteins downstream of TOR
are 4E binding protein-1 (4EBP1) and S6kinase 1 (S6K1),
and phosphorylation of both of these leads to stimula-
tion of protein synthesis and hence muscle hypertrophy.
54 PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org
know how this occurs, because subjects at risk of devel-
oping type 2 diabetes appear to have a relative deficit in
oxidative capacity (74, 83, 92). The first evidence that
the mechanism might involve AMPK came from stud-
ies in which the AMPK-activating drug AICAR was
found to cause upregulation of mitochondrial enzymes
in rat muscle (118). A transcriptional coactivator that is
a key regulator of mitochondrial biogenesis is PPAR-␥
coactivator-1␣ (PGC-1␣) (126). Intriguingly, mRNA
encoding PGC-1␣ is upregulated in rat epitrochlearis
muscle by low-intensity swimming exercise in vivo or
by AICAR treatment in vitro (111). These results sug-
gested that AMPK may be the sensor involved in the
mechanism that detects a deficit in oxidative capacity
and switches on mitochondrial biogenesis, a hypothe-
sis that is discussed further below. The mechanism by
which AMPK upregulates PGC-1␣ expression remains
unknown.
Downloaded from physiologyonline.physiology.org on October 27, 2010
The mechanism by which AMPK activation upregu-
lates GLUT4 also remains unclear, although it has
recently been reported that for myocyte enhancer fac-
tor-2 (MEF-2) and GLUT4 enhancer factor (GEF) [two
transcription factors required for GLUT4 expression
(88)], both the nuclear content and the binding to DNA
were increased in response to AICAR treatment of rats in
vivo (51), In the same study it was reported that AMPK
phosphorylates GEF in cell-free assays, although the site
was not identified and there is no evidence that this
occurs in vivo.
As well the ability of AMPK to stimulate muscle glu-
cose uptake as discussed earlier, it also appears to
stimulate fatty acid uptake. Thus incubation of rat car-
diomyocytes with AICAR, or the ATP synthase
inhibitor oligomycin, activated AMPK and increased
fatty acid uptake due to translocation of the FAT/CD36
transporter to the membrane (75). AMPK therefore
appears to stimulate both uptake and oxidation of fatty
acids, at least in cardiac muscle.
Although the muscle isoform of glycogen synthase
(GS) was shown to be phosphorylated by AMPK in
cell-free assays over 15 years ago (19), evidence that it
is a physiological target has only emerged recently.
AMPK phosphorylates GS at site 2 [Ser7 (19)], which
causes inactivation of the enzyme but also primes
phosphorylation at the neighboring site 2a (Ser10) by
casein kinase-1, which causes further inactivation (31,
130). Effects of phosphorylation at these sites on activ-
ity are overridden by high concentrations of glucose-
6-phosphate. One of us (D. G. Hardie) recently made
phosphospecific antibodies that recognize GS phos-
phorylated at site 2, or at sites 2 + 2a, and these have
now been used to show that AICAR treatment of
mouse muscle leads to phosphorylation of site 2 and,
to a lesser extent, sites 2 + 2a, with a concomitant inac-
tivation of the enzyme. Moreover, these effects are lost
in muscles from mice (described further in the next
section) in which the ␣2 isoform of AMPK has been
knocked out (62). These results strongly suggest that

GS is a physiological target for AMPK in skeletal mus-
cle. At first sight they appear to run counter to other
findings that muscle GS is activated rather than inacti-
vated during exercise, an effect that requires the func-
tion of the glycogen-bound form of protein phos-
phatase-1 (8). However, the level of glycogen appears
to have a dominant effect on the phosphorylation state
of GS, and activation of GS during exercise could be
due to glycogen depletion and might mask any under-
lying effect of AMPK. This interpretation is supported
by studies of patients with McCardle’s syndrome, a
hereditary defect in glycogen phosphorylase. Since
these subjects cannot break down muscle glycogen,
the effect of glycogen depletion on GS activity is lost,
and in these subjects exercise causes a marked activa-
tion of AMPK and inactivation of GS, as opposed to the
activation of GS seen in control subjects (86).
Physiological Role of AMPK in
Muscle: Studies with Transgenic
Mice
The most convincing evidence that AMPK is required
for the metabolic changes that occur in response to
endurance exercise comes from gene-targeting stud-
ies in mice. Global knockouts of both catalytic subunit
genes of AMPK (␣1 and ␣2) have been constructed
(63, 115). In the ␣2 knockout mice, but not the ␣1
knockout mice, the effects of AICAR on glucose
uptake, and on expression of mRNAs encoding PGC-
1␣ and HKII in skeletal muscle, were completely abol-
ished, although surprisingly the effects of contraction
or exercise on these processes were not (63, 64). At first
sight this suggests that these effects of contraction are
not mediated by AMPK, but a caveat with this
approach is that there is clearly some redundancy
between the ␣1 and ␣2 isoforms of AMPK, and double
knockouts have not yet been studied. An additional
complication is that in muscle from ␣2 knockout mice
the expression of ␣1 is upregulated twofold in skeletal
muscle (63, 115), which may provide some compensa-
tion for the lack of ␣2.
A possible route around this problem of redundan-
cy came with the development of mice in which mus-
cle AMPK was downregulated by expression of an
inactive, dominant negative mutant of the ␣2-subunit
from a strong promoter [muscle creatine kinase
(MCK)] (84). Although mRNA encoding the ␣2 mutant
was overexpressed 50-fold in skeletal muscle, the
expression of total ␣-subunit protein was unchanged
and the endogenous, active ␣-subunits appeared to
have been entirely replaced by the inactive mutant.
The explanation for this behavior may be that the ␣-
subunits are unstable in the absence of ␤ and ␥, so that
the level of expression of ␣-subunits is limited by the
availability of ␤- and ␥-subunits. Interestingly, the
effects of AICAR and hypoxia on glucose uptake were
completely eliminated in these mice, but the effects of
REVIEWS
contraction were only partially abolished, suggesting
that AMPK contributes to the latter but is not the
whole story (84). However, an important caveat with
this dominant negative approach concerns whether
the expression of the endogenous protein is complete-
ly eliminated. The Western blots in the original paper
indicate that the loss of the endogenous ␣-subunits
was substantial, but ␣1 and ␣2 were not assayed inde-
pendently and this did not rule out the possibility that
a small residual activity remained. Indeed, in another
paper using the same mice to study cardiac muscle
(where the MCK promoter is also expressed), ␣l activ-
ity remained unchanged (97). In a second mouse line
in which the inactive ␣2 subunit was expressed from a
cardiac muscle-specific (myosin heavy chain) promot-
er, a careful study of AMPK activity showed that ␣2
activity was reduced but not completely abolished,
whereas ␣1 activity was unaffected (127).
Downloaded from physiologyonline.physiology.org on October 27, 2010
Presumably because of their metabolic defects, the
mice expressing the dominant negative AMPK in
skeletal and cardiac muscle took much less voluntary
exercise (84). For this reason, it was not possible to
perform studies of the long-term effects of exercise
training in these mice. As a surrogate for this, the mice
were fed ␤-guanidinopropionic acid (GPA), a creatine
analog that depletes muscle ATP by interfering with its
replenishment from phosphocreatine. Feeding GPA
mimics many of the effects of endurance exercise in
skeletal muscle, including induction of PGC-1␣ and
mitochondrial biogenesis, but these effects were abol-
ished in the dominant negative mice (132). Thus, at
least when using GPA as a surrogate for endurance
exercise, AMPK appears to be essential for the upregu-
lation of mitochondrial biogenesis.
A third approach that our laboratories recently
introduced (99) is a muscle-specific knockout of the
upstream kinase LKB1 using Cre-loxP technology. An
advantage of this approach is that there is only one
LKB1 gene, eliminating problems of redundancy
when knocking out individual AMPK ␣-subunits. A
global LKB1 knockout is an embryonic lethal mutation
(129), but Ashworth’s laboratory created a line of mice
with loxP sites in the LKB1 gene. In these “floxed” mice
the expression and activity of LKB1 was already
reduced in skeletal muscle by ~90%, whereas the activ-
ities of the ␣1- and ␣2 isoforms of AMPK were reduced
by ~60% (99). This fortuitous effect of the loxP sites on
LKB1 expression turned out to be advantageous,
because the deletion of genes in skeletal muscle using
the Cre-loxP system is not always complete. Myocytes
are formed by fusion of many precursor cells, and if
the recombination induced by Cre recombinase has
failed in some of the precursors, residual expression of
the target gene may remain. By starting with a “floxed”
strain where the expression of LKB1 was already
decreased by 10-fold, and crossing with a deleter
strain that expressed Cre recombinase from a MCK
promoter, this resulted in a total loss of LKB1 activity
PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org 55
REVIEWS
and expression in skeletal muscle (99). The activity of
the ␣2 isoform of AMPK and its stimulation by con-
traction, AICAR or phenformin was also abolished.
Surprisingly, the low level of ␣1 activity in the “floxed”
mice was not reduced further on crossing with the
deleter strain, indicating either that this residual ␣1
was derived from nonmuscle cells (e.g., fibroblasts,
endothelial cells) or that there is an alternate
upstream kinase for ␣1. At least in cardiac muscle, the
latter explanation seems to be correct, because there is
a significant activity of the ␣1 isoform even in isolated
cardiac myocytes prepared from the “floxed” mice
expressing Cre recombinase, which are completely
deficient in LKB1 (100). These results show that LKB1
is absolutely required for the activity of the ␣2 isoform
in skeletal muscle and for full activation of ␣1, but the
possibility of an alternate upstream kinase for the ␣1
isoform in skeletal muscle requires further investiga-
tion.
Significantly, the effects of AICAR and contraction
on glucose uptake were normal in muscle from the
“floxed” mice (in which AMPK activity was reduced by
60%) but were greatly reduced in muscles from “floxed”
mice crossed with the deleter strain (in which ␣2 activ-
ity was completely abolished). This shows that the
LKB1
AMPK cascade is the primary signaling path-
way causing the increased glucose uptake in response
to contraction. However, in neither the mice expressing
the dominant negative AMPK mutant (84) nor the
LKB1 muscle knockouts (99) were the effects of con-
traction on glucose uptake completely eliminated,
which suggests that another signaling pathway also
functions in this process. Holloszy’s laboratory have
provided evidence that in rat muscle a Ca2+-mediated
pathway, possibly involving calmodulin-dependent
protein kinase II (CaMKII), is also involved (125).
CaMKII has also been found to be activated by cycle
ergometer exercise in human muscle (96).
In muscle from the mice in which LKB1 expression
was completely eliminated, phosphorylation of acetyl-
CoA carboxylase in response to AICAR or contraction
was also almost abolished, confirming that AMPK is the
kinase mainly responsible for phosphorylation of this
target, and the consequent stimulation of fatty acid oxi-
dation during exercise. Another interesting finding with
muscle from these mice was that the increases in the
ADP:ATP and AMP:ATP ratios induced by contraction
were much larger than those seen with muscle from the
wild-type controls. This provides direct genetic evi-
dence for the idea, first suggested many years ago (22),
that AMPK protects cells against stresses that deplete
ATP.
One caveat with the studies of the LKB1 knockout
mice is that LKB1 also acts upstream of several other
AMPK-related protein kinases (73), one or more of
which could also be required for the effects of contrac-
tion on glucose uptake. However, this seems rather
unlikely, because although several of the AMPK-relat-
56 PHYSIOLOGY • Volume 21 • February 2006 • www.physiologyonline.org
ed kinases are expressed in skeletal muscle, none of
them, other than AMPK, are activated by contraction
(98). Nevertheless, we recently found that the activity
of several of them, including QSK and MARK4, were
profoundly reduced in mice with a muscle-specific
knockout of LKB1, both in skeletal (3) and heart (K.
Sakamoto, J. Boudeau, and D. Alessi, unpublished
observation) muscle. Thus we cannot completely rule
out the possibility that these kinases are involved in
some of the metabolic effects of the LKB1 signaling
pathway.
Conclusions and Perspectives
The studies with knockout mice described in the pre-
vious section, particularly those using muscle-specific
LKB1 knockouts (99), suggest that AMPK is the “most
valuable,” although not the only, player determining
Downloaded from physiologyonline.physiology.org on October 27, 2010
the increased glucose uptake, fatty acid oxidation, and
inhibition of glycogen synthesis in skeletal muscle
during exercise, hence maintaining cellular energy
homeostasis. Thus many of the acute adaptations of
muscle to exercise appear to be mediated by this sys-
tem. Given that mice with defects in AMPK seem to be
intolerant of exercise (84), it may be more difficult to
study longer-term, chronic effects, such as the
increased mitochondrial biogenesis. However, the
studies using feeding of ␤-GPA to mice expressing the
dominant negative mutant of AMPK (132) already sug-
gest that the pathway may be crucial in those effects as
well. This reinforces the idea that AMPK signaling
pathway may be a good target for development of
drugs aimed at treatment of obesity and type 2 dia-
betes. Given that existing agents like metformin
appear to work indirectly by inhibiting mitochondrial
respiration, the AMP-binding domains on the ␥-sub-
units represent attractive new targets for drug devel-
opment.

D. G. Hardie is currently supported by a Programme
Grant from the Wellcome Trust (65565) and an Integrated
Project from the European Commission (QLG1-CT-2001-
01488). K. Sakamoto is supported by a grant from
Diabetes UK to Dario Alessi.
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