AMP-activated protein kinase in contraction regulation of ...

REVIEW

AMP-activated protein kinase in contraction regulation of

skeletal muscle metabolism: necessary and/or sufficient?

T. E. Jensen, J. F. P. Wojtaszewski and E. A. Richter

Molecular Physiology Group, Copenhagen Muscle Research Centre, Section of Human Physiology, Department of Exercise and

Sport Sciences, University of Copenhagen, Copenhagen, Denmark

Received 14 November 2008,

accepted 16 December 2008

Correspondence: E. A. Richter,

Molecular Physiology Group,

Copenhagen Muscle Research

Centre, Section of Human

Physiology, Department of

Exercise and Sport Sciences,

University of Copenhagen,

Copenhagen, Denmark.

E-mail: [email protected]

Abstract

In skeletal muscle, the contraction-activated heterotrimeric 5¢-AMP-acti-

vated protein kinase (AMPK) protein is proposed to regulate the balance

between anabolic and catabolic processes by increasing substrate uptake and

turnover in addition to regulating the transcription of proteins involved in

mitochondrial biogenesis and other aspects of promoting an oxidative

muscle phenotype. Here, the current knowledge on the expression of AMPK

subunits in human quadriceps muscle and evidence from rodent studies

suggesting distinct AMPK subunit expression pattern in different muscle

types is reviewed. Then, the intensity and time dependence of AMPK acti-

vation in human quadriceps and rodent muscle are evaluated. Subsequently,

a major part of this review critically examines the evidence supporting a

necessary and/or sufficient role of AMPK in a broad spectrum of skeletal

muscle contraction-relevant processes. These include glucose uptake,

glycogen synthesis, post-exercise insulin sensitivity, fatty acid (FA) uptake,

intramuscular triacylglyceride hydrolysis, FA oxidation, suppression of

protein synthesis, proteolysis, autophagy and transcriptional regulation of

genes relevant to promoting an oxidative phenotype.

Keywords AMPK, contraction, exercise, metabolism, skeletal muscle.

In skeletal muscle, AMP-activated protein kinase

(AMPK) signalling regulates the balance between

anabolic and catabolic processes by increasing sub-

strate uptake and turnover in addition to increasing

transcription of proteins involved in mitochondrial

biogenesis (Hardie 2008). Targeting AMPK in condi-

tions of nutrient oversupply and physical inactivity is

therefore an attractive strategy in the prevention of

metabolic diseases like type 2 diabetes and obesity

(Hardie 2007). This review will briefly discuss the

expression of AMPK in different muscle types, its

activation by exercise and proposed roles and targets

in the regulation of selected skeletal muscle processes.

For further information, the reader is referred to other

recent reviews on AMPK and exercise (Jørgensen et al.

2007a, Jørgensen & Rose 2008, Koh et al. 2008,

McGee & Hargreaves 2008, Witczak et al. 2008,

Richter & Ruderman 2009).

Main features of the AMPK trimer complex

The functional AMPK hetero-trimeric complex consists

of one of two catalytic a AMPK subunits in combina-

tion with one of two non-catalytic b AMPK subunits

and one of three c AMPK subunits (Hardie & Sakamoto

2006). The a subunits contain the kinase domains and

the functional heterotrimer requires phosphorylation at

Thr172 by upstream kinases for full activation (>100 ·increase in activity) (Corton et al. 1994, Weekes et al.

1994, Salt et al. 1998). Within the individual muscle

fibres, a2 AMPK has been reported in both the cytosol

and nucleus, while a1 seems expressed exclusively in the

Acta Physiol 2009, 196, 155–174

� 2009 The AuthorsJournal compilation � 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.01979.x 155

cytosol (Salt et al. 1998). The b subunits function as

scaffold proteins and interact with both the a and

c subunits (Woods et al. 1996, Hudson et al. 2003, Iseli

et al. 2005). In addition, b subunits contain an evolu-

tionally conserved glycogen-binding domain, which

allows AMPK to interact with glycogen particles

(Hudson et al. 2003, Polekhina et al. 2003, McBride

& Hardie 2009). In the c subunits, two AMP or ATP

molecules interact interchangeably with the interfaces

between the four tandem repeat cystathione b synthase

(CBS) motifs, while a third site binds AMP non-

exchangeably (Xiao et al. 2007). Via these interactions,

an increase in AMP/ATP ratio is relayed into in a

modest (less than fivefold) increase in AMPK trimer

activity (Corton et al. 1994). In addition, AMP binding

to the c subunits renders AMPK a poorer substrate for

its phosphatase, probably protein phosphatase (PP)2Ca(Davies et al. 1995, Sanders et al. 2007), enabling

upstream kinases to increase the phosphorylation of

AMPK aThr172 and cause further activation. Given

that the AMP/ATP ratio changes roughly in proportion

to the square of ADP/ATP ratio (Hardie & Hawley

2001), the sensing of this ratio makes AMPK an

exquisitely sensitive sensor of metabolic disturbances.

The molecular regulation of AMPK is summarized in

Figure 1a. For more in-depth discussion of structural

features of AMPK, see Towler & Hardie (2007),

Carling et al. (2008) and Bright et al. (2009).

Upstream kinases

In the yeast Saccharomyces cerevisiae, there are three

upstream kinases phosphorylating the activating

Thr210 site of the yeast AMPK orthologue, sucrose

non-fermenting (Snf)1 (Hong et al. 2005). It is therefore

no surprise that more than one AMPK Thr172 kinase

(AMPKK) has been identified in the more complex

mammalian setting. Of these, the ability of LKB1 and

Ca2+/calmodulin-dependent protein kinase kinases

(CaMKKs) to act as AMPKKs in various cell and tissue

types is now well established (Hawley et al. 2005,

Hurley et al. 2005, Woods et al. 2005). In addition,

transforming growth factor-b-activated kinase 1

(TAK1) is a direct AMPKK in vitro (Momcilovic et al.

Figure 1 Skeletal muscle AMPK expression and regulation. (a) Molecular regulation of AMPK activity. (b) AMPK expression

in human quadriceps and rodent oxidative and glycolytic muscles and their tentative activation profiles during contraction/exercise.

See text for details.

156� 2009 The Authors

Journal compilation � 2009 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2009.01979.x

AMPK and muscle metabolism in exercise Æ T E Jensen et al. Acta Physiol 2009, 196, 155–174

2006) but has also been proposed as an upstream

regulator of LKB1 in the heart (Xie et al. 2006). Finally,

a few studies in various non-muscle cell types have

suggested the phosphoinositide-3 kinase-related kinase,

Ataxia telangiectasia mutated (ATM), to be an AMPKK

during stimulation with insulin-like growth factor

(IGF)1 or anticancer drugs targeting double-stranded

DNA topoisomerase II (Suzuki et al. 2004, Sun et al.

2007, Fu et al. 2008). The potential relevance of the

latter two to skeletal muscle AMPK activation is yet to

be investigated.

In skeletal muscle, studies in two independent mouse

models lacking LKB1 have shown this kinase to be a

major skeletal muscle AMPKK (Sakamoto et al. 2005,

Koh et al. 2006). Both models display a striking

absence of a2 AMPK phosphorylation following stim-

ulation with the AMP mimetic compound aminoimi-

dazole-4-carboxymide-1-b-d-ribofuranoside (AICAR)

or intense contraction in situ (Sakamoto et al. 2005,

Thomson et al. 2007b) or ex vivo (Koh et al. 2006),

while a1 AMPK activity is only partially affected. Both

CaMKKa and b are detectable in a skeletal muscle

lysate, although they are expressed at very low levels

compared with that in the brain (Rose et al. 2006,

Jensen et al. 2007b). A study by our group using the

CaMKK inhibitor, STO-609, showed a potent inhibi-

tion of AMPK phosphorylation after 2 min of mild

tetanic contraction ex vivo but less so after 10 min,

suggesting that an AMPKK switch between CaMKK

and LKB1 may occur during exercise (Jensen et al.

2007b). Another study in mice did not observe

inhibition of AMPK activation by STO-609, perhaps

explained by the more intense contraction protocol

and/or lower STO-609 concentration used in that

study (Witczak et al. 2007). However, consistent with

CaMKK as a skeletal muscle AMPKK, that study

reported that overexpression of CaMKKa in skeletal

muscle by electroporation increases both a1 and a2

AMPK activity in mouse tibialis anterior (TA) muscle

(Witczak et al. 2007). Furthermore, overload of mouse

plantaris muscle by removal of gastrocnemius and

soleus muscles at the Achilles tendon (synergist-abla-

tion) in LKB1 knockout (KO) mice causes a marked

increase in the CaMKK expression and a1 AMPK

activity in skeletal muscle lysates (McGee et al.

2008a). Although CaMKKa overexpression increases

both a1 and a2 AMPK activity and STO-609 does not

affect AICAR-stimulated AMPK signalling ex vivo at

the 5 lm concentration used in Jensen et al. (2007b),

the observation that STO-609 inhibits both a1 and a2

AMPK activity at 2 min of contraction raises some

concerns regarding the specificity of STO-609, as a2

AMPK activity is clearly dependent on the presence of

LKB1 in mouse muscle (Sakamoto et al. 2005). Our

group is currently conducting experiments in muscles

from CaMKKa and b KO mice in an attempt to verify

the CaMKK effect suggested by STO-609.

AMPK subunit and trimer composition in

different fibre types

a1 AMPK is the predominant catalytic isoform in various

blood cells, endothelial cells, smooth muscle cells, nerve

and fat cells, which may contribute to AMPK protein

content measured in a whole muscle lysate. Therefore, it

is important to note that the muscle-specific expression of

a dominant negative kinase-dead (KD) AMPK construct

or LKB1 KO both nearly prevent a2 AMPK activity and

partially suppress a1 AMPK activity (Mu et al. 2001,

Fujii et al. 2005, Sakamoto et al. 2005, Koh et al. 2006,

Simard-Lefort et al. 2008), showing the expression of

both catalytic isoforms in skeletal muscle fibres. Of the

regulatory subunits, c3 AMPK has been reported to be

exclusively expressed in the fast-twitch glycolytic

extensor digitorum longus (EDL) mouse muscle but not

in the slow-twitch oxidative soleus mouse muscle (Barnes

et al. 2004). In addition, b1 AMPK has been observed to

be the major a2 AMPK-associated subunit in rat soleus,

while both b1 and b2 AMPK associate with a2 AMPK in

EDL muscle (Chen et al. 1999). This may explain some of

the observed differences in the activation profile of a1 and

a2 AMPK-containing complexes when comparing

slow-twitch oxidative rodent muscles with fast-twitch

glycolytic muscle types (Ai et al. 2002, Jensen et al.

2007b). An interesting report in porcine muscle described

a1 AMPK exclusively localized in skeletal muscle fibres

positive for type I MHC (Huber et al. 2007), suggesting

that the muscle differences in AMPK subunit expression

in rodents could even extend down to the muscle fibre

level. However, this finding should be followed up by

studies verifying the antibodies used by showing loss of

signal in a KO or knockdown model.

The AMPK–trimer composition in human quadriceps

resembles that of rodent glycolytic muscle in exhibiting

predominantly b2-associated AMPK activity and

expressing c3 AMPK (Birk & Wojtaszewski 2006).

Given that the human quadriceps is by far the predom-

inant muscle used for human biopsy studies and the

rodent studies indicate differences in trimer composition

between different muscles and/or fibre types, the com-

position and activation profile in other human muscles

require investigation.

Contraction activation of AMPK

Activation of total AMPK (without discriminating

between the different isoforms) in muscle during exer-

cise in rats was first shown by Winder & Hardie (1996).

It was later shown that in vivo exercise in rodents can

activate both the a1 and a2 AMPK-containing trimers in


Acta Physiol 2009, 196, 155–174 T E Jensen et al. Æ AMPK and muscle metabolism in exercise

various hind limb muscles (Jørgensen et al. 2005,

2007b), although a1 AMPK activation is not always

observed, even with presumably more intense in situ

nerve stimulation (Vavvas et al. 1997, Musi et al.

2001b, Sakamoto et al. 2005, Klein et al. 2007). Using

ex vivo contraction, a1 AMPK is more sensitive to

activation than a2 AMPK in mouse soleus and rat

epitroclearis muscle during low-intensity short-duration

contraction, while both isoforms are activated with

prolonged exercise/higher intensity (Hayashi et al.

2000, Barnes et al. 2002, Jørgensen et al. 2004a, Fujii

et al. 2005, Toyoda et al. 2006, Jensen et al. 2008). The

same a1 AMPK-specific activation profile is seen with

caffeine stimulation of SR-Ca2+ release below the

contraction threshold and is prevented by the ryanodine

receptor Ca2+ release inhibitor, dantrolene or the

CaMKK inhibitor, STO-609 (Jensen et al. 2007a),

hinting at a sarcoplasmic reticulum Ca2+, CaMKK-

dependent AMPK activation. A similar activation

pattern is seen when mouse quadriceps is subjected to

mild tetanic contractions in situ, suggesting that this is

not an ex vivo-specific phenomenon (T.E. Jensen &

E.A. Richter, unpublished data).

In human quadriceps muscle (i.e. vastus lateralis),

exercise at intensities above approx. 50–60% Vo2 peak

and varying duration have been routinely observed to

increase the activity of a2 AMPK-containing complexes

(Chen et al. 2000, Fujii et al. 2000, Wojtaszewski et al.

2000, Musi et al. 2001a, Nielsen et al. 2002, Stephens

et al. 2002). At the opposing end of the spectrum,

resistance exercise, characterized by intermittent con-

tractions of very high intensities, significantly activates

a2 AMPK in human quadriceps, although not as

potently as prolonged moderate-intensity exercise

(Dreyer et al. 2006). Examined in more detail using

sequential immunoprecipitation of trimer complexes,

very high intensity exercise (30 s of all-out sprint

exercise) increases a2b2c3 AMPK activity, while the

a2b2c1 activity decreases slightly (Birk & Wojtaszewski

2006). At a lower intensity/longer duration (67% Vo2

max, 90 min), the a2b2c3 activity increases progres-

sively to about 30-fold at 30 min of exercise and

remains around this level, while the a2b2c1 AMPK

activity increases slowly and steadily being about

threefold higher after 90 min (Treebak et al. 2007). In

contrast, activation of a1 AMPK complexes following

exercise in human quadriceps muscle is more contro-

versial with some having observed increased a1 AMPK

activity at both moderate-intensity exercise (Chen et al.

2003, Roepstorff et al. 2004, McConell et al. 2005) and

with 30 s of all-out sprint exercise (Chen et al. 2000),

while most studies observe no changes in a1 AMPK

activity at moderate intensities (Fujii et al. 2000,

Wojtaszewski et al. 2000, 2002b, 2003, Stephens et al.

2002) and even declining activity of a1b2c1 AMPK

complexes during 30 s of all-out sprint exercise (Birk &

Wojtaszewski 2006). For a summary of AMPK

expression and activation in human and rodent muscles,

see Figure 1b.

Effects of AMPK activation

Stimulation of glucose uptake

Mice carrying the R70Q c1 and R225Q c3 AMPK

mutations (hereafter referred to as c AMPK mutant

mice) are generally believed to have increased basal

AMPK activity (Barnes et al. 2004, Barre et al. 2007,

Garcia-Roves et al. 2008) (whether the R225Q c3

AMPK is genuinely an activating mutation has been

questioned; Yu et al. 2006). Both c AMPK mutations

are insufficient to increase basal glucose uptake, despite

a doubling of a1 and a2 AMPK-associated activity in

the c1 AMPK mutant mice and a presumed increase in

c3 AMPK mutant-associated activity. This suggests that

AMPK activation is not sufficient to increase glucose

uptake in skeletal muscle and the latter requires at least

one other necessary but AMPK-independent signal. This

proposal conflicts with studies in muscle cell culture,

where the expression of a constitutive active AMPK is

sufficient to increase glucose uptake (Fryer et al. 2002).

Meanwhile, a difference in sufficiency between models

is not unprecedented, as, e.g. rapid drug-inducible Akt

activation appears sufficient for full GLUT4 transloca-

tion in 3T3-L1 adipocytes (Ng et al. 2008) but not in L6

myotubes (Randhawa et al. 2008). However, the inter-

pretation of data obtained from the c AMPK mutant

mice is complicated by the massive glycogen accumu-

lation in these models. Still, the c3 AMPK mutant mice

show a clear increase in mitochondrial biogenesis in

mouse EDL muscle (Garcia-Roves et al. 2008), showing

that the AMPK pool is still sufficient to drive this

process while having no effect on basal glucose uptake.

A necessary (see below), but not sufficient, role of

AMPK activation in increasing glucose uptake would

explain the reported increases in a1 AMPK activity

without increased glucose uptake, for instance, in a2

AMPK KO muscles treated with AICAR (Jørgensen

et al. 2004a,b).

In ex vivo-incubated mouse muscles lacking various

AMPK-signalling proteins, activity of a2 AMPK, c3

AMPK and LKB1 is necessary for AICAR-stimulated

glucose uptake (Barnes et al. 2004, Jørgensen et al.

2004a,b, Sakamoto et al. 2005). It should be noted

that the lack of a2 AMPK prevents AICAR-stimulated

uptake in both mouse soleus and EDL muscle (Jørgen-

sen et al. 2004a,b), while the lack of AICAR-stimu-

lated glucose uptake in the c3 AMPK KO has only

been reported in mouse EDL (Barnes et al. 2004). As

mouse soleus muscles contain very little c3 AMPK, one




might speculate that a different c subunit combines

with a2 AMPK in soleus to signal the increase in

glucose uptake.

In relation to contraction, three studies from inde-

pendent groups using mice overexpressing a muscle-

specific dominant negative KD AMPK a2 subunit in

muscle, have independently confirmed that AMPK

catalytic activity is necessary to increase contraction-

stimulated glucose uptake ex vivo (Mu et al. 2001,

Jensen et al. 2007b, Simard-Lefort et al. 2008). Exper-

imental conditions have, furthermore, been defined in

which either a1 (low-intensity short-duration twitch

contraction; Toyoda et al. 2006, Jensen et al. 2008) or

a2 AMPK (AICAR, mitochondrial uncoupling) (Barnes

et al. 2004, Jørgensen et al. 2004a,b, Fujii et al. 2005,

Sakamoto et al. 2005) are necessary to increase glucose

uptake. Because ex vivo glucose uptake measurements

are transport limited (Hansen et al. 2000), these studies

favour a necessary role of AMPK in GLUT4 transloca-

tion ex vivo and, indeed, decreased GLUT4 transloca-

tion following AICAR and contraction stimulation has

been directly demonstrated in KD AMPK muscle by Mu

et al. (2001). In a mouse model distinct from but similar

to the KD AMPK mice overexpressing a dominant

negative kinase-dead a2 AMPK construct in muscle (a2i

mice), Goodyear’s group demonstrated that a partial

reduction in glucose uptake during intense ex vivo

contraction in the a2i EDL compared with wild-type

was absent, when lowering the voltage given to wild-

type EDL muscles ex vivo to match a decreased force

output in the a2i muscles (Fujii et al. 2005). Meanwhile,

this method lowers force output by decreasing fibre

recruitment and compares recruitment of all fibres in

the a2i muscles to fewer fibres recruited in wild-type,

making interpretations very difficult. In the KD AMPK

mice, force output ex vivo is essentially normal at low-

intensity tetanic contraction (1 s/15 s, 10 min) or pulse

frequencies below 50 Hz (Jensen et al. 2007b, 2008,

Simard-Lefort et al. 2008), yet glucose uptake is still

significantly lower in the KD AMPK muscles. The

difference between the a2i and KD AMPK models may

relate to differences in mouse strain (KD AMPK: C57Bl/

6, a2i: FVN) or contraction intensity. Low-intensity

protocols could be implemented in the a2i mice in an

attempt to settle the discrepancies between the two

models.

Puzzlingly, studies from our laboratory suggest that

the KD AMPK mice do not exhibit impaired glucose

uptake in vivo at either the same relative or absolute

running intensity (S.J. Maarbjerg, S.B. Jørgensen,

A.R. Rose, J. Jeppesen, T.E. Jensen, J.T. Treebak,

J.F.P. Wojtazewski and E.A. Richter, unpublished data).

Given the clear reductions in contraction-stimulated

glucose uptake ex vivo in this mouse model, this is a

rather surprising finding. The lack of difference in

glucose uptake does not appear to relate to a shift in the

rate-limiting step in vivo from transport by GLUT4 to

intracellular phosphorylation by hexokinase (Fueger

et al. 2004) as GLUT4 translocation assessed by

subcellular fractionation was also found to be normal

in the KD AMPK hind limb muscles (S.J. Maarbjerg,

S.B. Jørgensen, A.R. Rose, J. Jeppesen, T.E. Jensen, J.T.

Treebak, J.F.P. Wojtazewski and E.A. Richter, unpub-

lished data). A straightforward proposal is that the

in vivo environment enables signalling which bypasses

the requirement for AMPK. Interestingly, in this regard,

an unidentified serum factor has been shown to be

required for various stimuli to increase submaximal

insulin-stimulated glucose uptake 3.5 h post-contrac-

tion in rat epitroclearis ex vivo (Gao et al. 1994, Fisher

et al. 2002). A similar scenario where a serum factor

activates signalling pathways to circumvent the need for

AMPK in contraction-stimulated glucose uptake in vivo

might be envisioned.

Recently, attention has been focused on one of

Akt2/PKB’s many substrates, the Akt substrate of

160 kDa (AS160), as a potential downstream conver-

gence point between contraction and insulin signalling

to GLUT4 translocation. AS160 is phosphorylated at

multiple sites by Akt, a2 AMPK and possibly other

kinases, including phorbol ester-activated kinases

(reviewed in Cartee & Wojtaszewski 2007, Sakamoto

& Holman 2008, Zaid et al. 2008). By strict definition

as an Akt substrate of �160 kDa, muscle AS160

covers 2 distinct fibre type-specific proteins recognized

by the phospho-Akt substrate (PAS) consensus se-

quence antibody, with Tre-2 USP6-BUB2-Cdc16-do-

main family member D4 (TBC1D4) predominating in

oxidative and TBC1D1 in glycolytic muscles (Taylor

et al. 2008). The two highly related Rab GTPase-

activating proteins (GAPs) are thought to prevent

GLUT4 exocytosis, but not endocytosis, in various

models by keeping Rab proteins in their GDP-bound

form. Insulin- or contraction-induced phosphorylation

reduces the Rab GAP activity, causes dissociation from

GLUT4 vesicles and disinhibits GLUT4 translocation

(Cartee & Wojtaszewski 2007, Sakamoto & Holman

2008, Zaid et al. 2008).

While the exact role of TBC1D1 in glycolytic

skeletal muscle is not yet clear, TBC1D4 could, based

on in vivo electroporation studies (Kramer et al.

2006b, 2007), be partially involved in AMPK’s pro-

posed effects on contraction- and insulin-stimulated

GLUT4 trafficking. Interestingly, the calmodulin

(CaM)-binding domain of TBC1D4 seems partially

required for contraction-stimulated but not insulin-

stimulated glucose uptake in vivo (Kramer et al. 2007).

However, the following observations complicate mat-

ters in relation to contraction-stimulated glucose

uptake and TBC1D4 and TBC1D1:



(1) AMPK probably acts on GLUT4 endocytosis in

cardiac muscle (Yang & Holman 2005), while

TBC1D4 mediates GLUT4 exocytosis in 3T3-L1

adipocytes (Eguez et al. 2005).

(2) K+ depolarization increases PAS phosphorylation in

L6 myoblasts, probably reflecting TBC1D4 but not

TBC1D1 (Chavez et al. 2008) and induces GLUT4

translocation (Thong et al. 2007). However, K+

depolarization stimulus reduces GLUT4 endocyto-

sis (Wijesekara et al. 2006) but not exocytosis, the

proposed TBC1D4-regulated process in 3T3-L1

adipocytes (Eguez et al. 2005).

(3) Short-duration twitch contraction in mouse

soleus increases glucose uptake but not TBC1D4

phosphorylation (Jensen et al. 2008).

(4) Contraction-stimulated glucose uptake is normal in

a2 and c3 AMPK KO EDL muscle despite reduced

TBC1D1 phosphorylation (Barnes et al. 2004,

Jørgensen et al. 2004a,b, Treebak et al. 2006).

(5) Wortmannin inhibits insulin, stretch- and contrac-

tion-activated Akt phosphorylation (Sakamoto

et al. 2002, 2003) and insulin- and contraction-

activated PAS phosphorylation in mouse EDL or

rat epitroclearis (Bruss et al. 2005, Kramer et al.

2006a), at concentrations, which only inhibit

insulin-stimulated glucose uptake.

(6) In rat epitroclearis muscle, PAS phosphorylation

remains elevated 4 h post-exercise, when glucose

uptake is no longer increased (Arias et al. 2007).

(7) In rat epitroclearis muscle, PAS phosphorylation

during prolonged twitch contraction is transient,

yet glucose uptake remains elevated during

contraction (Funai & Cartee 2008).

(8) In humans, moderate-intensity exercise activates

glucose uptake within minutes of commencing

exercise (Wojtaszewski et al. 2003), while PAS

phosphorylation does not increase for the first

hour (Treebak et al. 2007).

Clearly, these findings question both the necessity and

sufficiency of TBC1D4 and TBC1D1 in regulating

contraction-stimulated glucose uptake and reveal

limitations in our current knowledge regarding

TBC1D4 and TBC1D1. It could be that TBC1D4

and TBC1D1 are recruited to different locations and

targets in response to different stimuli, which may

allow them to target exocytosis during insulin stim-

ulation and endocytosis during contraction. Moreover,

the PAS antibody does not recognize all eight phos-

phorylation sites on TBC1D4 and may miss important

phosphorylation changes induced by AMPK or other

proteins (Geraghty et al. 2007). A similar argu-

ment can be made for TBC1D1, which contains a

similar number of phosphorylation sites (Taylor et al.

2008).

AMPK activation and skeletal muscle glycogen

a2 AMPK has been proposed to phosphorylate glycogen

synthase (GS) on Ser7 (site 2) in skeletal muscle

(Jørgensen et al. 2004a), causing inactivation during

contraction. Therefore, it is perhaps surprising that

chronic skeletal muscle AMPK activation increases

skeletal muscle glycogen (Aschenbach et al. 2002,

Barnes et al. 2004, Barre et al. 2007, Costford et al.

2007), while some AMPK-deficient mice have 20–50%

lower baseline skeletal muscle glycogen (Mu et al.

2001, Koh et al. 2006). The exact mechanism behind

this relationship is not known but seems to involve

impaired glycogen synthesis as suggested in the c3

AMPK KO mice (Barnes et al. 2004). The higher

glycogen in c AMPK mutant mice may relate to a push

effect by AMPK-stimulated glucose uptake driving an

allosteric activation of GS by glucose-6-phosphate.

While this explanation is possible, lower, not higher,

basal glucose uptake is observed in the c1 and c3 mutant

AMPK mice, and basal glucose uptake is not lower in

AMPK-deficient mouse models. Importantly, skeletal

muscle glycogen can increase independent of increased

glucose uptake as seen in the muscle-specific GLUT4

KO mice, where glycogen is doubled despite an 80%

reduction in basal glucose uptake (Kim et al. 2005). In

these mice, the PP1-targeting regulatory subunits RGL/

GM and protein targeting to glycogen (PTG) were

increased by 300–400% compared with that in

wild-type, hinting at possible mechanisms behind the

increased glycogen synthesis. Whether AMPK regulates

PP1-targeting subunit expression or function has, to our

knowledge, not been investigated.

Some studies have found an inverse correlation

between pre-exercise muscle glycogen concentrations

and the degree of AMPK activation in both rats and

humans (Derave et al. 2000, Wojtaszewski et al. 2002a,

2003, McConell et al. 2008). Mouse muscles contain

roughly 25–50% of the glycogen content seen in rats

(Mu et al. 2001, Jørgensen et al. 2004b) and 5–10% of

the glycogen content found in humans (Wojtaszewski

et al. 2003). Lower skeletal muscle glycogen content in

rodents compared with that in humans may contribute

to the faster activation of AMPK in the rodent models.

In human muscle, glycogen-depletion increases the basal

activity of both a1 and a2 AMPK trimers in resting

muscle and causes a more rapid and higher activation of

a2 AMPK trimers during bicycle exercise (Wojtaszewski

et al. 2003). However, a causal relation between low

AMPK activity and high glycogen content is uncertain.

In a human training study, where 10 days of exercise

training was followed by an exercise/diet intervention to

elicit a 50% difference in skeletal muscle glycogen, the

suppressive effect of training on a1 and a2 AMPK




activation was similar between groups, suggesting that

the suppression of AMPK activity may not relate

directly to glycogen content (McConell et al. 2005).

Decreased activation of a2 AMPK-containing trimers

may in part relate to a decreased c3 AMPK expression

following exercise training (Nielsen et al. 2003, Frøsig

et al. 2004, Wojtaszewski et al. 2005). Furthermore,

glycogen phosphorylase-defective McArdle’s disease

patients exhibit an exaggerated AMPK activation with

exercise despite greatly elevated skeletal muscle glyco-

gen, which may, however, relate to a higher metabolic

stress in the exercise-intolerant McArdle’s patients

(Nielsen et al. 2002). Obviously, the inverse relation-

ship between skeletal muscle glycogen and AMPK

activation requires further study.

Interaction with insulin-stimulated glucose

uptake

AMPK activation has long been proposed to mediate

the increase in glucose uptake at a given submaximal

insulin concentration (insulin sensitivity) commonly

observed in the hours post-exercise (Richter et al.

1982, Wojtaszewski & Richter 2006). Fisher et al.

(2002) observed in rat epitroclearis muscle that AMPK-

activating stimuli, such as hypoxia, AICAR, ex vivo

contraction and swimming exercise, elevated submax-

imal insulin-stimulated glucose uptake compared with

that in control, when studied 3.5 h later at which time

the effects of these stimuli on basal glucose uptake were

no longer present. As mentioned earlier, the increase

seems to depend on a still unidentified serum protein

known to be >10 kDa and distinct from insulin, IGF-I,

serum albumin or a, b or c-globulin (Gao et al. 1994).

Subsequently, it was shown in the same model, that

p38 mitogen-activated protein kinase (MAPK) activa-

tion with anisomycin also increases submaximal insu-

lin-stimulated glucose uptake in a manner preventable

by the p38 MAPK inhibitor SB203580, while the

contraction-stimulated increase in insulin sensitivity

is not sensitive to SB203580 (Geiger et al. 2005).

Importantly, AMPK does not seem to signal through

p38 MAPK in mouse skeletal muscle (Ho et al. 2007),

strongly suggesting that multiple pathways can increase

insulin sensitivity post-stimulation in skeletal

muscle. In fact, even stimulation of glucose uptake

with insulin itself increases submaximal insulin-stimu-

lated glucose uptake 3.5 h later in rat muscles (Geiger

et al. 2006).

From studies in the AMPK-deficient mouse models,

the interaction of AMPK activation with insulin sensi-

tivity is not clear. a2 AMPK KO mice are insulin

resistant at the whole-body level, but both a1 and a2

AMPK KO soleus and EDL muscles have normal

submaximal insulin-stimulated glucose uptake ex vivo

(Viollet et al. 2003). Maximal insulin-stimulated glu-

cose uptake is not reduced in EDL muscles from the KD

AMPK animals (Mu et al. 2001), but maximal insulin

stimulation does not reflect insulin sensitivity. In soleus

muscles from 36-week-old a2i mice (Fujii et al. 2008),

submaximal insulin-stimulated glucose uptake tends to

be lower (50%) in chow-fed animals compared with

that in wild-type (although this tendency is not apparent

in younger animals; Fujii et al. 2007). Consistent with a

role of AMPK in regulating insulin sensitivity, feeding

these mice a high-fat diet for 30 weeks partially reduces

insulin-stimulated glucose uptake by wild-type soleus

muscles, while completely abrogating it in a2i soleus

muscles (Fujii et al. 2008). By contrast, submaximal

insulin-stimulated glucose uptake in soleus muscles

ex vivo is not affected in LKB1 KO mice compared

with that in wild-type, while LKB1 KO EDL muscles

display increased Akt Thr308 phosphorylation and

submaximal insulin-stimulated glucose uptake, perhaps

because of reduced expression of the direct endogenous

Akt-inhibitor, Tribbles 3 (Koh et al. 2006).

As mentioned in the previous section, TBC1D4 and

TBC1D1 are both AMPK substrates. Swimming

exercise in rats causes an increase in the PAS phosphor-

ylation 4 h post-exercise (Arias et al. 2007), probably

reflecting both TBC1D4 and TBC1D1 (Funai & Cartee

2008). A similar sustained increase in PAS phosphory-

lation has been reported in humans 3 h after 60 min of

moderate-intensity bicycle exercise (Howlett et al.

2008). Being activated at the time of increased insulin

sensitivity, both TBC1D4 and TBC1D1 are attractive

candidates in increasing insulin sensitivity. Interestingly,

our laboratory has observed in humans that TBC1D4

phosphorylation on Ser318, Ser341, Ser588, Ser751 but

not Ser641, the primary site recognized by the PAS

antibody (Geraghty et al. 2007), is increased 4 h post-

exercise and further increased by insulin (Treebak et al.

2009).

Lipid metabolism

AMPK has been proposed to be involved in the

regulation of fatty acid (FA) uptake in skeletal muscle,

as AICAR infusion in rats increased skeletal muscle FA

uptake (Shearer et al. 2004). Furthermore, it has been

demonstrated that AICAR-stimulated FA uptake is

severely blunted in mice that do not express fatty acid

transporter (FAT)/cluster of differentiation (CD)36

(Bonen et al. 2007), indicating a major role of this

putative sarcolemmal FA transport protein in AICAR-

stimulated palmitate uptake and oxidation in skeletal

muscle. Thus, AMPK may be part of the signal

mediating AICAR- and contraction-stimulated translo-

cation of CD36 and FA uptake, analogous to the

proposed role of AMPK in promoting GLUT4 translo-



cation. However, some studies have clearly demon-

strated a discrepancy between FA uptake and AMPK

activation during low-intensity contraction in the

perfused rat hind limb (Raney et al. 2005), as well as

between AMPK activation and CD36 translocation

(Turcotte et al. 2005), suggesting additional mecha-

nisms regulating FA uptake in skeletal muscle.

Skeletal muscle fibres contain intramuscular triacyl-

glyceride (IMTG) which can be mobilized as FA for

ATP production by the action of two specific lipases,

hormone-sensitive lipase (HSL) and adipose triacyl-

glycerol lipase (ATGL) (Watt & Steinberg 2008).

Among the lipases, the activity of the diacylglycerol

(DAG) lipase HSL is increased in response to contrac-

tion of rodent and human muscle in a transient and

intensity-independent fashion (Langfort et al. 2000,

Watt et al. 2003, 2004, Roepstorff et al. 2004). Global

HSL KO mice display DAG accumulation and

decreased FFA release in skeletal muscle but no

difference in IMTG (Haemmerle et al. 2002). AMPK

phosphorylates HSL on Ser565, proposed to counter-

act b-adrenergic stimulation of HSL activity during

prolonged exercise (Watt et al. 2004), although others

have found Ser565 to have no bearing on HSL activity

in human skeletal muscle (Roepstorff et al. 2004).

Consistent with the inhibition of HSL and IMTG

mobilization by AMPK, AICAR stimulation reduces

IMTG hydrolysis in rodent soleus muscle (Muoio et al.

1999, Smith et al. 2005). Based on a recent report in

the KD AMPK mice, AMPK seems required for

AICAR-stimulated but not contraction-stimulated

HSL Ser565 phosphorylation (Dzamko et al. 2008).

In addition to HSL, the skeletal muscle TG lipase,

ATGL, may play an important role in the mobilization

of skeletal muscle FA stores. Hence, global ATGL KO

approximately reduces the TG hydrolase activity by

half in skeletal muscle and causes massive triglyceride

accumulation in multiple tissues including skeletal

muscle (Haemmerle et al. 2006). Recently, it was

reported that significant ATGL activity is present in

human skeletal muscle and that expression in muscle is

increased by endurance training (Alsted et al. 2009).

Whether ATGL is regulated by AMPK has not been

investigated.

Once taken up by skeletal muscle or released from

IMTG stores, the oxidation of FA to generate ATP has

been proposed to be regulated in part by allosteric

inhibition of the mitochondrial FA transporter carnitine

palmitoyltransferase (CPT)1 by malonyl-CoA (Winder

et al. 1990, 1995, Winder & Hardie 1996, Saha et al.

1997, Vavvas et al. 1997). Indeed, malonyl-CoA

decreases markedly in rodent skeletal muscle during

both fasting and contraction, commensurate with an

increase in FA oxidation (McGarry et al. 1983, Winder

et al. 1990, 1995, Winder & Hardie 1996, Hutber et al.

1997, Vavvas et al. 1997), whereas a correlation

between malonyl-CoA and fat oxidation is not always

evident in humans during exercise (Odland et al. 1996,

1998, Roepstorff et al. 2005). Acetyl-CoA carboxylase-

2 (ACC2), the mitochondrial membrane enzyme pro-

ducing malonyl-CoA from acetyl-CoA, is allosterically

activated by citrate and inhibited by palmitoyl-CoA and

covalently inhibited by phosphorylation on Ser221

(human site orthologous to rat Ser218 and mouse

Ser212; http://www.phosphosite.org/siteAction.do?id=

39279, Trumble et al. 1995, Winder & Hardie 1996,

Saha et al. 1997, Vavvas et al. 1997, Winder et al.

1997). ACC2 Ser221 is arguably the best described

AMPK site in skeletal muscle during AICAR and

contraction stimulation (Mu et al. 2001, Fujii et al.

2005, Sakamoto et al. 2005, Koh et al. 2006, Simard-

Lefort et al. 2008) and is routinely used as a read-out of

endogenous AMPK activity. Global ACC2 KO in mice

markedly reduces malonyl-CoA and increases FA

oxidation, presumably driving the lean, hyperphagic

phenotype seen in these mice (Abu-Elheiga et al. 2001).

While the marked disinhibition of FA oxidation in

ACC2 KO mice demonstrates its importance to fat

oxidation and validity as a drug target (Abu-Elheiga

et al. 2003, Choi et al. 2007), the necessity of AMPK in

the regulation of ACC2 and FA oxidation is controver-

sial. Thus, muscle-specific LKB1 KO mice display

normal baseline FA oxidation and remain capable of

phosphorylating ACC2 and decreasing malonyl-CoA in

response to in situ contraction (Thomson et al. 2007a).

On the other hand, these mice display attenuated

AICAR-stimulated decreases in malonyl-CoA and

impaired AICAR-stimulated fat oxidation in EDL

muscle (contraction not investigated). The KD AMPK

mice exhibit partially reduced AICAR and contraction-

stimulated ACC2 phosphorylation in some but not all

muscles, and do not exhibit any defect in basal, AICAR-

or contraction or exercise-stimulated FA oxidation ex

vivo or in vivo (Dzamko et al. 2008). Whether residual

AMPK activity in the KD AMPK mice or non-AMPK

kinases rescues the fat oxidation under these conditions

remains to be elucidated. Using a bioinformatics

approach to screen 190 kinases in skeletal muscle from

wild-type and KD AMPK mice contracted in situ,

extracellular signal-regulated kinase 1/2, MKK3/6 and

inhibitor of jB kinase a/b showed the largest genotype

difference in phosphorylation (Dzamko et al. 2008).

However, in vitro kinase assays against recombinant

ACC2 suggests that neither of these is ACC2 kinases

(Dzamko et al. 2008). Importantly, a lack of correlation

between AMPK activity and fat oxidation is observed in

both rodents (Raney et al. 2005) and humans (Chen

et al. 2000, McConell et al. 2005, Roepstorff et al.

2005, 2006), supporting that there must be physiolog-

ical alternatives to AMPK in regulating fat oxidation.




Therefore, AMPK-mediated inhibition of ACC2 is

probably sufficient to increase fat oxidation in skeletal

muscle but not necessary.

The degradation of malonyl-CoA is regulated by

malonyl-CoA decarboxylase (MCD) (Dyck et al. 1998).

As expected, global MCD KO mice exhibit lower fat

oxidation and higher malonyl-CoA, as do cultured

human myotubes with silenced MCD (Bouzakri et al.

2008, Koves et al. 2008). Although MCD is activated

by both AICAR and contraction stimulation in rat

muscle (Saha et al. 2000, Park et al. 2002), MCD does

not appear to be a substrate of AMPK (Habinowski

et al. 2001), again suggesting the involvement of other

kinases in its regulation.

Protein synthesis

Consistent with its role as an energy homeostat, AMPK

has been shown to inhibit signalling through the

anabolic mammalian target of rapamycin (mTOR)

pathway to protein synthesis by phosphorylation of

tuberous sclerosis (TSC)2 Thr1227 and Ser1345 of the

TSC1/TSC2 Rheb GAP complex (Inoki et al. 2003),

mTOR Thr2446 (Cheng et al. 2004) in addition to the

mTOR complex 1 protein, Raptor, on Ser722 and

Ser792, causing sequestration by 14-3-3 (Gwinn et al.

2008). When ex vivo insulin stimulation or in situ

contraction in rodent EDL muscles is preceded by

AICAR treatment, the phosphorylation of the mTOR

substrates S6 kinase (S6K)1 and 4E-binding protein

(4E-BP)1 is suppressed (Deshmukh et al. 2008,

Thomson et al. 2008a), suggesting that AMPK inhibits

contraction and insulin-stimulated mTOR signalling. In

agreement, human resistance exercise also activates

AMPK, correlating with suppressed 4E-BP1 phosphor-

ylation and protein synthesis (Dreyer et al. 2006). The

AICAR effect appears to be at least partially dependent

on AMPK, as a2 and c3 AMPK KO EDL muscles lose

part of the suppression of insulin-stimulated S6K1 and

4E-BP1 phosphorylation (Deshmukh et al. 2008). The

incomplete disinhibition of insulin-stimulated mTOR

signalling in the a2 and particularly c3 AMPK KOs in

that study may be due to rescue by remaining AMPK

subunits or non-AMPK signalling pathways. Impor-

tantly, the lack of suppressive effect on protein synthesis

seems to translate into increased fibre size in some of the

AMPK-deficient mouse models as soleus and EDL

muscles from KD AMPK mice have been reported to

have 5% and 20% greater cross-sectional areas than

wild-type (Mu et al. 2003). Surprisingly, mice lacking

LKB1 in muscle do not show a similar exaggerated

hypertrophy in plantaris muscle at baseline or following

4 weeks of overload by synergist ablation compared

with wild-type (McGee et al. 2008a). At baseline, the

low number of animals in that study (n = 4 per group)

combined with the use of muscle weight to evaluate

hypertrophy may not have been sensitive enough to

detect genotype differences <20%. A lack of exagger-

ated hypertrophy in LKB1 KO muscles may reflect

compensation by increased CaMKK or increased TAK1

activation acting through a1 AMPK, as proposed by the

authors. Compensation by a1 AMPK is consistent with

TSC2 phosphorylation at the AMPK site Ser1345

increasing normally with overload in LKB1 KO muscle

(McGee et al. 2008a). However, overload activation of

AMPK in wild-type or LKB1 KO muscles is clearly not

sufficient to suppress S6K or 4E-BP1 phosphorylation

induced by synergist ablation, possibly because this

represents a sufficiently strong stimulus to overcome

any inhibitory effects of AMPK. Protein synthesis is

decreased in human skeletal muscle during both

dynamic (Rennie 2005) and resistance exercise (Dreyer

et al. 2006). The role of AMPK in regulating protein

synthesis during exercise is, however, uncertain. Thus,

Thr56 phosphorylation of eukaryotic elongation factor

2 (eEF2), which inhibits translation, occurs very rapidly

and independently of exercise intensity in humans (Rose

et al. 2008), suggesting regulation by Ca2+ rather than

AMPK. Furthermore, inhibition of protein synthesis as

well as increased eEF2 and 4E-BP1 phosphorylation

following ex vivo contractions is similar in wild-type

and KD AMPK muscles (Rose et al. 2009), suggesting a

minor role of AMPK in depressing skeletal muscle

protein synthesis during exercise.

Protein breakdown

The degradation of cellular material is accomplished by

two mechanisms: the multi-enzyme ubiquitin–protea-

some system and autophagy, the engulfment of cellular

components in autophagosomes and their subsequent

fusion with lysosomes (Mammucari et al. 2008).

Studies in cell culture and mice have suggested that

AMPK may control ubiquitin conjugation and prote-

asomal degradation during muscle atrophy. In C2C12

myotubes, AICAR, metformin or 2-deoxyglucose

increases myofibrillar degradation in addition to mRNA

expression of atrogin-1/muscle atrophy F-box (MAFbx)

and muscle-specific RING finger protein (MuRF)1

(Krawiec et al. 2007, Nakashima & Yakabe 2007).

These enzymes are E3 ubiquitin ligases important to

ubiquitin–proteasome degradation during muscular

atrophy (Bodine et al. 2001, Gomes et al. 2001). The

increases in atrogin-1/MAFbx and MuRF1 mRNA with

the aforementioned stimuli are preventable by the

AMPK inhibitor compound C (Krawiec et al. 2007,

Nakashima & Yakabe 2007). Furthermore, AICAR

injection in mice induces atrogin-1/MAFbx and MuRF1

mRNA in muscle (Krawiec et al. 2007). This probably

involves increased AMPK-dependent expression



(Nakashima & Yakabe 2007) and activation (Greer

et al. 2007) of the Forkhead box O3 (FoxO3)

transcription factor, a key mediator of autophagy

(Mammucari et al. 2008). Autophagy has also been

shown to depend on AMPK in the yeast S. cerevisiae

(Wang et al. 2001). Furthermore, transfection

experiments with a KD AMPK construct or with

constitutively active AMPK construct in various cell

culture systems suggest a major, necessary but

non-sufficient role in stimulating autophagy (Meley

et al. 2006). Similar to atrogin-1/MAFbx and MuRF1

regulation, increased expression of key genes in the

autophagic/lysosomal pathway including light chain

(LC)3 and BCL2/adenovirus E1B-interacting protein

(Bnip)3 appear to involve inhibition of mTOR signal-

ling and activation of FoxO3 (Krawiec et al. 2007,

Mammucari et al. 2007, Zhao et al. 2007), both known

effects of AMPK activation (Greer et al. 2007; studies

mentioned above).

Control of oxidative muscle phenotype by

AMPK

By regulating the activity of a number of transcription

factors, AMPK has been implicated in many of the

phenotypic changes in muscle seen with endurance

training, including fibre-type shift from type IIx to IIa

and mitochondrial biogenesis. As an example, chronic

treatment of rodents with AICAR or the phospho-

creatine depleting agent, b-guanadinopropionic acid,

activates AMPK and increases a number of key mito-

chondrial enzymes in addition to mitochondrial content

in skeletal muscle (Winder et al. 2000, Zhou et al.

2000, Bergeron et al. 2001, Bamford et al. 2003,

Putman et al. 2003). AMPK activity is a requirement

to increase mitochondrial content with these stimuli as

the effect of b-guanadinopropionic acid is absent in the

KD AMPK mice (Zong et al. 2002) and AICAR has no

effect on mitochondrial marker enzymes in a2 AMPK

KO muscles (Jørgensen et al. 2007b). Meanwhile, a

necessary role of AMPK in mitochondrial biogenesis in

response to exercise training is controversial. a2 AMPK

KO and muscle-specific LKB1 KO mice display training

adaptations in mitochondrial enzyme proteins similar to

wild-type despite a decreased running ability and a

general reduction in many mitochondrial enzymes

(Jørgensen et al. 2007b, Thomson et al. 2007b). This

probably reflects redundant signalling pathways regu-

lating endurance-type phenotypic changes in skeletal

muscle. As a testament to this, overexpression of active

forms of calcineurin (Ryder et al. 2003, Long et al.

2007), PKD (Kim et al. 2008), MAPK kinase (MKK)6

(the p38 MAPK kinase) (Akimoto et al. 2005) and

CaMKIV (Wu et al. 2002) (not normally expressed in

skeletal muscle; Akimoto et al. 2004b, Rose et al. 2006)

in addition to overexpression of downstream transcrip-

tion factors like MEF2 (Potthoff et al. 2007) or PGC1a(Lin et al. 2002) are all sufficient to increase key

markers of mitochondrial biogenesis. Regardless of this,

sufficiency is the more relevant criterion in terms of

AMPK as a drug target, and mitochondrial markers are

increased in skeletal muscles of mice overexpressing the

AMPK-activating c1 or c3 mutant isoforms (Nilsson

et al. 2006, Rockl et al. 2007, Garcia-Roves et al.

2008), showing that AMPK activation is sufficient to

induce endurance-type adaptations.

At the molecular level, transcriptional regulation by

AMPK involves a multitude of cytosolic and nuclear

targets. Among its substrates, the transcriptional

co-activator, PGC1a seemingly orchestrates many of

the endurance exercise-like changes seen with AMPK

activation (Scarpulla 2002, Handschin & Spiegelman

2008). Muscle-specific PGC1a overexpression increases

mitochondrial biogenesis and fatigue resistance in mice

(Lin et al. 2002), while muscle-specific PGC1a KO mice

display a general reduction in metabolic gene expression

and exercise intolerance resembling features described

in AMPK-deficient mice (Mu et al. 2001, Handschin

et al. 2007, Jørgensen et al. 2007b, Thomson et al.

2007b). Surprisingly, despite a general reduction in key

metabolic enzymes, PGC1a KO mice, like AMPK-

deficient mice, remain capable of increasing these

enzymes in response to exercise training (Leick et al.

2008).

AMPK activation is sufficient to increase PGC1amRNA in c3 AMPK mutant mouse muscle (Garcia-

Roves 2008). This may involve direct PGC1aphosphorylation by AMPK on Thr177 and Ser538,

phosphorylations required to induce PGC1a auto-tran-

scription (Jager et al. 2007). In addition, elegant elec-

troporation studies of mouse TA muscle with PGC1apromoter mutants have shown that both the two MEF-

binding sites and the CRE-binding site in the PGC1apromotor are required for contraction-stimulated

PGC1a transcription (Akimoto et al. 2004a, 2008).

Because AMPK activation is sufficient to increase

PGC1a transcription (Garcia-Roves et al. 2008), it

seems probable that AMPK directly or indirectly regu-

lates MEF and CRE-dependent PGC1a promoter tran-

scription. Indeed, AICAR treatment in rats has been

shown to induce MEF2 translocation to the nucleus

without detectable phosphorylation of MEF2 (Holmes

et al. 2005). Meanwhile, another transcription factor,

GLUT4 enhancer factor (GEF), appears to be directly

phosphorylated by AMPK and is imported to the

nucleus in response to AICAR treatment, and this event

temporally precedes the import of MEF2 (Holmes et al.

2005). Increased DNA binding of both MEF2 and GEF

is seen with 60 min of bicycling exercise in humans

(McGee et al. 2006). As MEF2A is known to interact




with GEF (Knight et al. 2003), it has been speculated

that GEFs aid in the nuclear import of MEF2 (Holmes

et al. 2005). In addition, both MEF and GEF have been

shown in 3T3-L1 adipocytes to interact with HDAC5,

another AMPK target which, upon phosphorylation on

Ser259 and Ser498, binds 14-3-3 proteins and is

exported from the nucleus (McGee et al. 2008b,

Sparling et al. 2008). In relation to the regulation of

the CRE site, CREB Ser133 phosphorylation is lower in

LKB1 KO muscles compared with that in wild-type and

partially purified AMPK from rat liver and skeletal

muscle is capable of phosphorylating CREB1, in addi-

tion to ATF1, CRE modulator (CREM) and CREB-like

2 (CREBL2), but not ATF2 (Thomson et al. 2008b).

Surprisingly, a recent study in C2C12 muscle cells found

that the AICAR-responsive region of the PGC1apromoter is a GATA/Ebox sequence interacting with

the transcription factor upstream stimulatory factor-1,

which indicates a permissive role of the MEF and CRE

sites (Irrcher et al. 2008). FoxO3 activation by AMPK is

probably also necessary to increase PGC1a transcrip-

tion, because FoxO3 KO mouse embryonic fibroblast

cells, expressing FoxO3 mutated at the 6 AMPK

phosphorylation sites, Ser399, Ser413, Ser555, Ser588

and Ser626, are not capable of increasing PGC1amRNA in response to 2-deoxyglucose treatment (Greer

et al. 2007). The mechanism by which FoxO3 increases

PGC1a mRNA could in part involve an increased

activity of the NAD+-dependent deacetylase, sirtuin

(SIRT)1, through transcription of the NAD+ biosyn-

thetic enzyme nicotinamide phosphoribosyltransferase

(Nampt)/Visfatin (Fulco et al. 2008). Active SIRT1

deacetylates PGC1a to increase some, but not all,

PGC1a-influenced transcription in addition to other

transcriptional cofactors and activators including MEF2

and FoxO (Brunet et al. 2004, Nemoto et al. 2005,

Rodgers et al. 2005, Zhao et al. 2005).

Conclusion

Figure 2 illustrates the different substrates of AMPK

discussed in this review and the proposed function of

these. Redundancy appears to be a recurring theme in

skeletal muscle contraction and AMPK signalling. Seen

in this light, the relatively mild phenotype of many of

the AMPK-deficient mouse models is not surprising.

Given the right (often reductionistic) experimental

circumstances, however, sufficient and occasionally

even essential roles of AMPK can be shown in AMPK

transgenic mouse models. An immediate challenge lies

in settling the apparent discrepancies between the

findings in the various AMPK transgenic mouse

models to reach consensus on the role of AMPK in

various aspects of skeletal muscle metabolism. How-

ever, the major obstacle in the future will most

certainly lie in investigating the sufficiency and/or

necessity of skeletal muscle AMPK in the more

relevant human setting.

Conflict of interest

There is no conflict of interest.

The study was supported by the Novo Nordisk Research

Foundation, The Danish Diabetes Association, an Integrated

Project (LSHM-CT-2004-005272) and COST action BM0602

GLUT4 translocation (contraction,insulin-interaction)

Oxidative

PGC1alpha HDAC5

GEF / MEF2

CREB family

FOXO3

phenotype GS

TBC1D4

Rab GTPases

TBC1D1 Glycogen

FA uptake

CD36?

HSL

ACC2 TSC1 / TSC2 Rheb Raptor

mTOR

4E-BP1 S6K1

Ubiquitin / proteasomal autophagic / lysosomal muscle breakdown

GTPase

FA oxidation

Muscle growth

IMTG hydrolysis

synthesis?

bb

a g

… …

nu

cleu

s

GbL

P P

P P

P P

P P

P P

P P

P P

P P P P

P P

P P

P P

P P

P P

P P

Figure 2 Summary of the AMPK substrates covered in this review and their proposed action in skeletal muscle. Red arrow:

inhibitory effect, green arrow: stimulatory effect. See text for details.



funded by the European Commission, The Lundbeck Founda-

tion, The Copenhagen Muscle Research Centre and The

Danish Medical and Natural Sciences Research Councils.

Jacob Jeppesen and Thomas Junker Alsted are thanked for

helpful comments in relation to AMPK regulation of lipid

metabolism.

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