Download Bypassing the glucose/fatty acid cycle: AMP

The Glucose/Fatty Acid Cycle 1963–2003
Bypassing the glucose/fatty acid cycle:
AMP-activated protein kinase
D. Carling1 , L.G.D. Fryer, A. Woods, T. Daniel, S.L.C. Jarvie and H. Whitrow
MRC Clinical Sciences Centre, Cellular Stress Group, Imperial College, Hammersmith Hospital Campus, DuCane Road, London W12 ONN, U.K.
Abstract
The AMPK (AMP-activated protein kinase) cascade plays a key role in regulating energy metabolism.
Conditions which cause a decrease in the ATP/AMP ratio lead to activation of AMPK. Once activated, AMPK
initiates a series of responses that act to restore the energy balance of the cell. In skeletal muscle, activation
of AMPK increases both glucose uptake and fatty acid oxidation, raising the possibility that AMPK can
bypass the glucose/fatty acid cycle. This review focuses on the role of AMPK in the regulation of glucose
and fatty acid metabolism in muscle. Recently, naturally occurring mutations within the γ isoforms have
been identified which lead to altered metabolic regulation in cardiac and skeletal muscle and suggest an
important role for the kinase in regulating glycogen metabolism.
Introduction
AMPK (AMP-activated protein kinase) is the central
component of a protein kinase cascade that plays an important
role in the regulation of energy metabolism [1,2]. AMPK
is a heterotrimeric enzyme that has been highly conserved
throughout evolution and homologues of all three subunits
have been identified in plants, yeast, nematode worms, flies
and mammals [1]. Studies in mammalian cells have shown that
AMPK is activated in response to conditions that deplete
ATP. Once activated, AMPK initiates a series of responses
aimed at restoring ATP levels. Two major consequences
of AMPK activation are switching-off of ATP-consuming
pathways and switching-on of ATP-generating pathways.
This review will concentrate on the role of AMPK in skeletal
and cardiac muscle, highlighting the important role AMPK
plays in regulating energy metabolism and discussing the
recent findings that mutations in the γ-subunit isoforms lead
to marked alterations in metabolism in these tissues.
Regulation of muscle metabolism by AMPK
Skeletal muscle plays a major part in determining whole-body
energy metabolism. In humans, skeletal muscle accounts for
over 70% of total body glucose disposal [3]. Moreover,
muscle is a versatile tissue in terms of its substrate utilization
and can use a number of fuels, including fatty acids. The
regulation of energy utilization in muscle, which is subject
to large fluctuations, e.g. during the change from rest to
contraction and back to rest again, requires tight control.
The first major clue to indicate that AMPK plays an
important role in regulating skeletal muscle energy metabolism came from studies in which rat muscle was perKey words: AMP-activated protein kinase, energy metabolism, exercise, fatty acid oxidation,
glucose uptake, metabolic syndrome.
Abbreviations used: ACC, acetyl-CoA carboxylase; AICA, 5-amino-4-imidazole carboxamide;
AMPK, AMP-activated protein kinase; CBS, cystathionine-β-synthase; ZMP, AICA riboside monophosphate.
To whom correspondence should be addressed (e-mail [email protected]).
1
fused with AICA (5-amino-4-imidazole carboxamide) riboside [4]. This compound is taken up by cells and
phosphorylated to the monophosphate form, ZMP (AICA
riboside monophosphate), which can accumulate to relatively
high levels within certain cell types. ZMP mimics the activatory effects of AMP on the AMPK cascade [5,6] and has
been used in numerous studies to investigate the physiological
consequences of AMPK activation. Perfusion of rat hindlimb
muscle with AICA riboside led to an increase in the rate
of fatty acid oxidation and an increase in glucose uptake
[4]. The increased rate of fatty acid oxidation correlated
with inactivation of ACC (acetyl-CoA carboxylase) and a
subsequent fall in the concentration of malonyl-CoA, the
end product of the reaction catalysed by ACC. There are two
isoforms of ACC, encoded by separate genes [7,8]. ACC1
(ACCα; molecular mass 265 kDa) is expressed primarily
in lipogenic tissues, such as liver, adipose and lactating
mammary gland, and produces malonyl-CoA for fatty
acid synthesis. ACC2 (ACCβ; 280 kDa) is predominantly
expressed in cardiac and skeletal muscle and malonyl-CoA
generated by this isoform influences the transport of fatty
acids into the mitochondria through inhibition of carnitinepalmitoyl-CoA transferase I, regulating the rate of fatty acid
oxidation [9]. A number of studies from several groups have
demonstrated that AMPK phosphorylates and inactivates
both of these isoforms [1]. Phospho-specific antibodies
have been developed to the major AMPK phosphorylation
site within ACC1 (Ser-79) and these cross-react with the
equivalent site within ACC2 (Ser-227 in human ACC2),
allowing a simple method for determining phosphorylation
of ACC by AMPK in tissues.
Recently, leptin was found to activate AMPK in skeletal
muscle, providing a molecular basis for the stimulatory effect
of this hormone on fatty acid oxidation [10]. The activation of AMPK by leptin showed a marked bi-phasic
response. A rapid (15-min) response is likely to be a direct
effect of leptin binding to receptors on the muscle cell. This
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is supported by the finding that leptin activates AMPK in
H-2Kb muscle cells in culture. In contrast, a slower (6-h)
response appears to be mediated via the hypothalamus and
stimulation of α-adrenergic receptors. The precise molecular
mechanism(s) by which leptin activates AMPK are not fully
understood, although there is evidence that at least part of
the action of leptin on AMPK may involve a nucleotideindependent pathway (see below). Whereas leptin appears to
increase fatty acid oxidation in skeletal muscle through activation of AMPK, this does not appear to be the case in cardiac
muscle [11]. This finding suggests that AMPK has tissuespecific effects and this is likely to be an important consideration when examining the physiological role of the kinase.
In addition to activating fatty acid oxidation, AICA
riboside perfusion of rat muscle led to an increase in glucose
uptake [4]. The increase in glucose transport induced by
AICA riboside was accompanied by increased translocation
of GLUT4 glucose transporters to the plasma membrane [12].
A number of studies have shown that AMPK is activated
in muscle in response to contraction, raising the possibility
that AMPK could mediate the effect of contraction on
glucose uptake [13]. In order to address this question directly
Birnbaum’s group [14] generated transgenic mice expressing a
dominant-negative form of AMPK in muscle. The activation
of glucose uptake in these mice by either AICA riboside or
hypoxia was completely abolished, whereas the activation
in response to contraction was partially inhibited. These
findings suggest that AMPK mediates the effect of AICA
riboside and hypoxia on glucose uptake, but there must
be at least two mechanisms involved in the contractionmediated increase in glucose uptake: an AMPK-dependent
and an AMPK-independent pathway. In H-2Kb muscle cells
activation of glucose uptake in response to AICA riboside or
hyperosmotic stress was completely blocked by adenoviral
expression of a dominant-negative form of AMPK [15].
In the same study, expression of a constitutively active
form of AMPK stimulated glucose uptake concomitant with
increased translocation of both GLUT1 and GLUT4 to
the plasma membrane [15]. Precisely how AMPK regulates
glucose transport remains enigmatic, although it is clear that
it is distinct from the insulin-mediated pathway which
involves stimulation of phosphoinositide 3-kinase [13].
Activation of AMPK
A key finding early on in the study of AMPK was the
observation that the kinase cascade was activated in response
to ATP depletion [16]. Since then many studies have
demonstrated the activation of AMPK following a rise in
the AMP/ATP ratio within the cell. The AMPK cascade is
activated by AMP via a number of separate mechanisms and
all of these are antagonized by high concentrations of ATP,
resulting in an ultrasensitive system responding to changes in
the AMP/ATP ratio [17]. More recently, however, evidence
has emerged that the AMPK cascade can also be activated in
response to stimuli that do not cause a detectable change in the
AMP/ATP ratio. Several studies have shown that metformin,
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a widely used oral anti-hyperglycaemic agent [18], activates
AMPK without altering cellular nucleotide levels [19–21].
In addition, hyperosmotic stress activates AMPK in H-2Kb
muscle cells without an increase in the AMP/ATP ratio [20].
The mechanism for the nucleotide-independent activation of
AMPK is not known, but it does involve phosphorylation
of threonine-172 within the catalytic α-subunit of AMPK, the
major site phosphorylated by the upstream kinase, AMPK
kinase [20,22]. Whether there are multiple upstream kinases
acting on AMPK in response to different stimuli, or whether
different signalling pathways converge on the same upstream
kinase, is unclear. Identification of the upstream kinase(s)
in the AMPK cascade will play a significant role in helping
elucidate this issue.
Mutations in the γ-subunit isoforms lead
to muscle abnormalities
Three γ-subunit isoforms of AMPK have been identified
[23] and all have a highly conserved C-terminal domain
that contains four copies of a motif known as a CBS
(cystathionine β-synthase) domain [24]. The function of
this domain, which is found in a wide variety of proteins,
including CBS itself, is not known, although there is evidence
to suggest that in AMPK these domains may be involved
in nucleotide binding [23]. A dominant mutation present in
Hampshire pigs that causes high glycogen content in skeletal
muscle was found to map to the γ3 gene [25]. A single amino
acid substitution changing Arg-200 to a Gln (R200Q) was
found to be uniquely associated with the affected allele [25].
Two potential initiating sites have been identified within
pig γ3, and the numbering used here refers to residues
downstream of the second initiating methionine, which was
the form that was originally reported for the pig γ3 sequence
[25]. The equivalent residue in human γ3, taken from the first
initiating methionine, is Arg-226 [23]. In humans, mutations
have been identified within γ2 that cause cardiac hypertrophy
associated with Wolff–Parkinson–White syndrome [26–29].
Intriguingly, some of the mutations identified in γ2 lie in
equivalent positions to the γ3 mutation within individual
CBS domains (Figure 1). The effects of four different
mutations within γ2 were studied by co-expressing γ2 with
α1β1 or α2β1 in mammalian cells [30]. Three of these
mutations occur within the CBS domains and had a significant
effect on regulation of the kinase by AMP. No direct effect on
activity was observed with a fourth mutation, corresponding
to the insertion of a leucine residue between two adjacent CBS
domains. These findings suggest that the CBS domains play
a role in AMP binding and that mutations which affect AMP
binding may have significant consequences for the normal
development and function of the heart. In addition, the
results also raise the possibility that distinct mutations leading
to the same disease phenotype have different effects on
AMPK activity, indicating that alternative mechanisms may
contribute to similar pathogenesis. In a separate study the effect of introducing a mutation into γ1 equivalent to the
R302Q mutation in γ2 or the R200Q mutation in γ3 was
The Glucose/Fatty Acid Cycle 1963–2003
Figure 1 Locations of naturally occurring mutations within the CBS domains of AMPK γ2 and γ3
The amino acid sequences of the CBS domains of human γ2, in which the R302Q (CBS1), H383R (CBS2) and R531G (CBS4)
mutations are located, are shown aligned. The CBS domain of pig γ3 (CBS1), which contains the R200Q mutation, is also
shown aligned. Amino acid identities in the CBS domains of the γ isoforms are shaded in black and conservative substitutions
in grey. The residues at which the mutations occur are denoted by arrows above the sequences.
determined [31]. In that study, the mutation was also found to
reduce the AMP dependence of the kinase, but in contrast
to γ2 [30] the mutation in γ1 was reported to cause
constitutive activation. At present it is unclear whether these
differences reflect differences in the γ isoforms, or whether
there are other factors that complicate the findings.
Unusual vacuoles, possibly containing glycogen, were
found to be present in cardiomyocytes from patients
harbouring two different mutations in γ2 (N488I and T400N)
[29]. This is particularly interesting, since the γ3 mutation
in pigs leads to high skeletal muscle glycogen content. Taken
together these results suggest that a common mechanism may
exist by which mutations in γ2 and γ3 lead to excess glycogen
accumulation in heart and skeletal muscle, respectively.
However, as mentioned above, it is possible that the different
mutations within the γ2-subunit do not have the same effect
on AMPK activity. Furthermore, the effects of the γ2 and
γ3 mutations appear to be restricted either to heart (γ2) or
skeletal muscle (γ3). In the case of γ3 this may simply reflect
the expression pattern of the protein which is confined almost
exclusively to skeletal muscle [23]. However, γ2 is expressed
in a number of tissues, raising the possibility that γ2 has a
specific function in heart and that changes in this function lead
to the disease phenotype. This seems an attractive hypothesis
given the finding that γ1 accounts for the majority of AMPK
activity in all tissues, including heart and skeletal muscle [23].
AMPK and glycogen
A recent observation which seems likely to feature strongly
in the regulation of AMPK is the discovery of a motif within
the β-subunit that appears to play a role in glycogen binding
[32,33]. This motif, originally known as an N-isoamylase
domain, is found in a number of enzymes, most of which are
involved in the metabolism of α1-6 branches in α1-4 glucans.
In the AMPK β-subunits there is evidence that this domain
binds glycogen and so it has been renamed a glycogen-binding domain [32,33]. At present the role of the glycogen-
Figure 2 Links between AMPK and glycogen
A model highlighting some of the potential links between AMPK and
glycogen metabolism is shown. (1) AMPK increases glucose uptake into
the cell; (2) AMPK phosphorylates and inactivates glycogen synthase
in vitro; (3) high glycogen suppresses activation of AMPK; (4) the βsubunit contains a glycogen-binding domain which could target AMPK
to glycogen. Refer to the text for more details.
binding domain in the β-subunit isoforms is not fully
understood although it is tempting to speculate that it will
be involved in the regulation of glycogen metabolism by
AMPK. Previously, AMPK has been shown to phosphorylate
and inactivate glycogen synthase in vitro [34] and high muscle
glycogen has been shown to suppress the activation of AMPK
in response to AICA riboside [35]. These combined findings
clearly implicate a complex link between AMPK activity, glycogen synthesis and the level of glycogen within the cell (Figure 2). How these different aspects linking AMPK to
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glycogen metabolism fit with the effect of γ-subunit mutations on glycogen accumulation in vivo remains to be
elucidated. The efforts of a number of groups working in
this area are likely to ensure that some of the pieces of the
jigsaw will fall into place in the near future.
References
1 Hardie, D.G., Carling, D. and Carlson, M. (1998) Annu. Rev. Biochem. 67,
821–855
2 Kemp, B.E., Mitchelhill, K.I., Stapleton, D., Michell, B.J., Chen, Z.-P. and
Witters, L.A. (1999) Trends Biochem. Sci. 24, 22–25
3 Ferrannini, E. (1998) Endocrinol. Rev. 19, 477–490
4 Merrill, G.F., Kurth, E.J., Hardie, D.G. and Winder, W.W. (1997) Am. J.
Physiol. 273, E1107–E1112
5 Sullivan, J.E., Brocklehurst, K.J., Marley, A.E., Carey, F., Carling, D. and
Beri, R.K. (1994) FEBS Lett. 353, 33–36
6 Corton, J.M., Gillespie, J.G., Hawley, S.A. and Hardie, D.G. (1995) Eur. J.
Biochem. 229, 558–565
7 Abu-Elheiga, L., Jayakumar, A., Baldini, A., Chirala, S.S. and Wakil, S.J.
(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4011–4015
8 Ha, J., Lee, J.-K., Kim, K.-S., Witters, L.A. and Kim, K.-H. (1996) Proc. Natl.
Acad. Sci. U.S.A. 93, 11466–11470
9 Ruderman, N.B., Saha, A.K., Vavvas, D. and Witters, L.A. (1999) Am. J.
Physiol. 276, E1–E18
10 Minokoshi, Y., Kim, Y.B., Peroni, O.D., Fryer, L.G., Muller, C., Carling, D.
and Kahn, B.B. (2002) Nature (London) 415, 339–343
11 Atkinson, L.L., Fischer, M.A. and Lopaschuk, G.D. (2002) J. Biol. Chem.
277, 29424–29430
12 Kurth-Kraczek, E.J., Hirshman, M.F., Goodyear, L.J. and Winder, W.W.
(1999) Diabetes 48, 1667–1671
13 Hayashi, T., Hirshman, M.F., Kurth, E.J., Winder, W.W. and Goodyear, L.J.
(1998) Diabetes 47, 1369–1373
14 Mu, J., Brozinick, J.T., Valladares, O., Bucan, M. and Birnbaum, M.J. (2001)
Mol. Cell. Biol. 7, 1085–1094
15 Fryer, L.G.D., Foufelle, F., Barnes, K., Baldwin, S.A., Woods, A. and Carling,
D. (2002) Biochem. J. 363, 167–174
16 Corton, J.M., Gillespie, J.G. and Hardie, D.G. (1994) Curr. Biol. 4, 315–324
17 Hardie, D.G., Salt, I.P., Hawley, S.A. and Davies, S.P. (1999) Biochem. J.
338, 717–722
C 2003
Biochemical Society
18 Stumvoll, M., Nurjhan, N., Perriello, G., Dailey, G. and Gerich, J.E. (1995)
N. Engl. J. Med. 333, 550–554
19 Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., Wu, M.,
Ventre, J., Doebber, T., Fujii, N. et al. (2001) J. Clin. Invest. 108,
1167–1174
20 Fryer, L.G., Patel, A.P. and Carling, D. (2002) J. Biol. Chem. 277,
25226–25232
21 Hawley, S.A., Gadalla, A.E., Olsen, G.S. and Hardie, D.G. (2002) Diabetes
51, 2420–2425
22 Hawley, S.A., Davison, M.D., Woods, A., Davies, S.P., Beri, R.K., Carling, D.
and Hardie, D.G. (1996) J. Biol. Chem. 271, 27879–27887
23 Cheung, P.C.F., Salt, I.P., Davies, S.P., Hardie, D.G. and Carling, D. (2000)
Biochem. J. 346, 659–669
24 Bateman, A. (1997) Trends Biochem. Sci. 22, 12–13
25 Milan, D., Jeon, J.T., Looft, C., Amarger, V., Robic, A., Thelander, M.,
Rogel-Gaillard, C., Paul, S., Iannuccelli, N., Rask, L. et al. (2000) Science
288, 1248–1251
26 Blair, E., Redwood, C., Ashrafian, H., Ostman-Smith, I. and Watkins, H.
(2001) Hum. Mol. Genet. 10, 1215–1220
27 Gollob, M.H., Green, M.S., Tang, A.S.L., Gollob, T., Karibe, A., Hassan, A.,
Ahmad, F., Lozado, R., Shah, G., Fananapazir, L. et al. (2001) N. Engl.
J. Med. 344, 1823–1831
28 Gollob, M.H., Seger, J.J., Gollob, T.N., Tapscott, T., Gonzales, O., Bachiniski,
L. and Roberts, R. (2001) Circulation 104, 3030–3033
29 Arad, M., Benson, D.W., Perez-Atayde, A.R., McKenna, W.J., Sparks, E.A.,
Kanter, R.J., McGarry, K., Seidman, J.G. and Seidman, C.E. (2002) J. Clin.
Invest. 109, 357–362
30 Daniel, T. and Carling, D. (2003) J. Biol. Chem. 277, 51017–51024
31 Hamilton, S.R., Stapleton, D., O’Donnell, J.B., Kung, J.T., Dalal, S., Kemp,
B.E. and Witters, L.A. (2001) FEBS Lett. 500, 163–168
32 Polekhina, G., Gupta, A., Michell, B.J. van Denderen, B., Murthy, S., Feil,
S.C., Jennings, I.G., Campbell, D.J., Witters, L.A., Parker, M.W. et al. (2003)
Curr. Biol. 13, 867–871
33 Hudson, E.R., Pan, D.A., James, J., Lucocq, J.M., Hawley, S.A., Green, K.A.,
Baba, O., Terashima, T. and Hardie, D.G. (2003) Curr. Biol. 13, 861–866
34 Carling, D. and Hardie, D.G. (1989) Biochim. Biophys. Acta 1012, 81–86
35 Wojtaszewski, J.F.P., Jørgensen, S.B., Hellsten, Y., Hardie, D.G. and
Richter, E.A. (2002) Diabetes 51, 284–292
Received 8 July 2003