Covalent activation of heart AMP-activated protein kinase in response
to physiological concentrations of long-chain fatty acids
Hilary Clark
1
, David Carling
2
and David Saggerson
1
1
Department of Biochemistry and Molecular Biology, University College London, UK;
2
Cellular Stress Group, MRC Clinical Sciences
Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK
Rat hearts were perfused for 1 h with 5 m
M
glucose with or
without palmitate or oleate at concentrations characteristic
of the fasting state. The inclusion of fatty acids resulted in
increased activities of the a-1 or the a-2 isoforms of AMP-
activated protein kinase (AMPK), increased phosphoryla-
tion of acetyl-CoA carboxylase and a decrease in the tissue
content of malonyl-CoA. Activation of AMPK was not
accompanied by any changes in the tissue contents of
ATP, ADP, AMP, phosphocreatine or creatine. Palmitate
increased phosphorylation of Thr172 within AMPK
a-subunits and the activation by palmitate of both AMPK
isoforms was abolished by protein phosphatase 2C leading
to the conclusion that exposure to fatty acid caused activa-
tion of an AMPK kinase or inhibition of an AMPK phos-
phatase. Invivo, 24 h of starvation also increased heart
AMPK activity and Thr172 phosphorylation of AMPK

[9,11] that covalent activation of the AMPK may also occur
through upstream processes independent of the ÔclassicalÕ
pathway, e.g. involving the LKB1 tumour-suppressor
kinase [12,13].
Malonyl-CoA has an important role in the regulation
of fuel selection by the heart [14,15] through its potent
inhibition [16] of carnitine palmitoyltransferase-1 (CPT1).
Malonyl-CoA is synthesized and disposed of by acetyl-CoA
carboxylase (ACC) and malonyl-CoA decarboxylase
(MCD), respectively. ACC is inactivated through phos-
phorylation by the AMPK [17–19]. By contrast, at present
there is conflicting evidence for or against the notion that
MCD can be activated following phosphorylation by the
AMPK [20–22]. Dyck and Lopaschuk [14] and Kudo et al.
[23] have shown during postischaemic reperfusion of the
rat heart that elevation of AMPK activity correlates with
decreased ACC activity, decreased malonyl-CoA content
andanincreasedrateofb-oxidation. Work from our
laboratory had shown that heart malonyl-CoA content was
increased by insulin [15,24] and insulin has been shown to
decrease AMPK activity in heart [7]. However, Awan and
Saggerson [15] and Hamilton and Saggerson [24] showed
that long-chain fatty acid (palmitate) both decreased
malonyl-CoA content and prevented the effect of insulin
to increase malonyl-CoA. Therefore we investigated the
effect of physiological concentrations of long-chain fatty
acids on AMPK activity in perfused rat hearts in the
expectation that AMPK activity would be increased. This
was found to occur through covalent modification of
AMPK a subunits driven by an unknown upstream protein

4
)GLHLVK] was from Upstate
Biotechnology. Recombinant phosphoprotein phosphatase
2C (PP2C; human a-isoform) was a generous gift from
R. Beri (AstraZeneca Pharmaceuticals). Sodium palmitate
or oleate were bound to fatty acid-poor BSA [26] and the
concentration of bound fatty acid was standardized with a
Wako NEFA test kit (Alpha Laboratories).
Animal procedures
1
Male Sprague–Dawley rats (300–350 g body weight) were
maintained at 20–22 °C on a 13 h light/11 h dark cycle with
light from 06:00 h to 19:00 h. Rats were anaesthetized with
sodium pentobarbitone (300 mgÆkg
)1
) injected intraperiton-
eally prior to removal of the heart. Hearts from fed animals
were perfused retrogradely via the aorta at 37 °Cwith
100 mL Krebs–Henseleit bicarbonate (KHB) medium
equilibrated with O
2
/CO
2
(19 : 1) containing 1.3 m
M
CaCl
2
,
5m
M

M
NaF, 5 m
M
Na
4
P
2
O
7
,1m
M
dithiothreitol, 1 m
M
phenyl-
methanesulfonyl fluoride, 1 m
M
benzamidine and soybean
trypsin inhibitor (4 lgÆmL
)1
). The homogenate was centri-
fuged at 4 °Cfor10minat13000g and 250 lLofthe
supernatant incubated for 2 h at 4 °C with anti-AMPK
serum (usually 15 lL) bound to Protein G-Sepharose.
Immunoprecipitates were collected by centrifugation (1 min
at 5200 g). Normally immunoprecipitates were washed/
recentrifuged once with 300 lL homogenization/immuno-
precipitation buffer and then twice (4 °C) with 300 lLof
AMPK assay buffer (40 m
M
Hepes pH 7.0 contain-

orthophosphoric acid (1%, v/v) and then twice for 10 min
in water before drying and scintillation counting. In
experiments with PP2C fresh immunoprecipitates (see
above) were washed with 300 lL homogenization/immu-
noprecipitation buffer and then twice (4 °C) with 300 lL
50 m
M
Tris/HCl pH 7.4 containing 1 m
M
dithiothreitol.
After recovery by centrifugation
2
(see above) these immu-
noprecipitates were resuspended in 25 lLof50m
M
Tris/
HClpH7.4,10m
M
MgCl
2
,1m
M
dithiothreitol, glycerol
(5%, v/v) and PP2C (160 lgÆmL
)1
). MgCl
2
was omitted
from control incubations. After 30 min at 30 °Cthe
immunoprecipitates were again recovered by centrifugation

M
Tris/HCl pH 7.5 containing 0.14
M
NaCl and 0.1%
(v/v) Tween 20 (NaCl/Tris). After blocking with a solution
of milk powder (5% w/v) for 1 h the membranes were
washed again in NaCl/Tris and then re-blotted with AMPK
a-subunit primary antibody (Cell Signalling Technology).
Metabolites
ATP, ADP and AMP were measured in neutralized
trichloroacetic acid extracts of frozen heart after separation
by HPLC [29] and creatine and phosphocreatine as
2 H. Clark et al. (Eur. J. Biochem.) Ó FEBS 2004
described [30,31]. Malonyl-CoA was measured as described
by Awan & Saggerson [24]. Perfusion media and rat serum
were assayed for non-esterified fatty acid (NEFA; Wako
test kit) and glycerol [32]. Glucose was measured in
haemolysed blood samples [33].
Statistics
Values are given as means ± S.E.M. Statistical significance
was calculated using Student’s t-test for paired or unpaired
samples as indicated.
Results
Long-chain fatty acids cause phosphorylation of
a-subunits and activation of AMPK in perfused heart
Perfused hearts were fuelled by 5 m
M
glucose alone or by
glucose with 0.075 m
M

palmitate net removal of NEFA from the
perfusate occurred (Table 2).
Unexpectedly we found that a-1 and a-2 AMPK activities
tendedtobelowestinheartsstartedwith0.075m
M
palmitate and showed a significant decrease relative to
control 1 when assays contained the allosteric effector AMP
(Table 1). The adult heart normally supports much of its
ATP production from fatty acid oxidation [34]. Therefore
the control 1 condition may be one of metabolic stress,
reflected by a higher AMPK activity state than under
normal fed conditions.
Using control 1 as the baseline, 0.25 or 0.5 m
M
palmitate increased a-1 and a-2 AMPK activities by at
least2-foldwhenAMPwasomittedfromtheseassays.
Activation of a-2 AMPK by these fasting concentrations
of NEFA was also seen in assays with AMP. By contrast,
with AMP present, a-1 AMPK appeared to be insensitive
to palmitate. In essence, covalent activation following
exposure to fatty acid and allosteric activation by AMP
were mutually exclusive effects for a-1 AMPK whereas for
Table 1. The effect of perfusion with long-chain fatty acids on the activity state of heart AMPK. Hearts were perfused for 60 min with 5 m
M
glucose,
BSA (20 mgÆml
)1
) and sodium palmitate or oleate as indicated. The values are means ± S.E.M. of the numbers of independent measurements
shown in parentheses.
Initial NEFA concentration

e
(6) 4.14 ± 0.42
c,h
(6) 5.54 ± 0.50
a,f,I
(11)
0.5 m
M
Palmitate 2.79 ± 0.37
c,h
(11) 2.95 ± 0.53
g
(11) 5.16 ± 0.55
d,h
(11) 8.44 ± 0.90
d,h,l
(11)
0.5 mm Oleate 2.52 ± 0.27
c,h
(5) 2.67 ± 0.33
g
(5) 4.61 ± 0.63
c,g
(5) 8.15 ± 1.28
b,g,k
(5)
a,b,c,d
P < 0.05, < 0.02, < 0.01, < 0.001 respectively versus zero NEFA (unpaired test).
e,f,g,h
P < 0.05, < 0.02, < 0.01, < 0.001

)1
Æg wet wtÆheart
)1
) [B]
Total fatty acid utilisation
(lmolÆh
)1
Æg wet wtÆheart
)1
)
[B–A]
Zero (control 1) 0.068 ± 0.003 +4.03 ± 0.60 7.03 ± 1.18 3.00
0.075 m
M
Palmitate
(control 2)
0.082 ± 0.005 +0.41 ± 0.38 6.73 ± 0.93 6.32
0.15 m
M
Palmitate 0.108 ± 0.005 )2.28 ± 0.34 5.22 ± 0.82 7.50
0.25 m
M
Palmitate 0.154 ± 0.005 )4.76 ± 0.28 5.88 ± 1.26 10.64
0.5 m
M
Palmitate 0.342 ± 0.016 )7.45 ± 1.22 5.45 ± 1.51 12.90
Ó FEBS 2004 Activation of AMPK by fatty acids (Eur. J. Biochem.)3
a-2 AMPK these appeared to be two independent effects
(Table 1).
Using control 2 as the baseline, perfusion with 0.25 or

and also recognizes the equivalent AMPK phosphorylation
site in ACC-2 (280 kDa). However after perfusion with
0.5 m
M
palmitate phosphorylation of both 265 and
280 kDa bands was clearly seen (Fig. 1). This was accom-
panied by a significant 51% decrease in malonyl-CoA
content (Table 3).
Figs 2 and 3 show experiments which support the
conclusion that activation of AMPK by fatty acid was
due to increased protein phosphorylation. Treatment of
immoprecipitates with PP2C abolished the activation due to
0.5 m
M
palmitate (Fig. 2). If Mg
2+
, which is required for
PP2C activity, was omitted the activation by palmitate was
not abolished (data not shown). The AMPK activities in
Fig. 2 are lower than those in Table 1 whilst the degree of
activation by palmitate was higher. The reason for this is
unclear but it is stressed that more extensive washing of
immunoprecipitates was necessary in order to remove
inhibitors of protein dephosphorylation before treatment
with PP2C. Fig. 3 shows that exposure of hearts to 0.5 m
M
palmitate significantly increased P-T172 abundance in the
combined AMPK a-1 and a-2 subunits by 2.5-fold without
causing any change in the abundance of AMPK a-subunit
protein.

)1
). The Ôenergy chargeÕ
was calculated from (ATP +
1
/
2
ADP)/(total adenine nucleotides) [35].
Perfusate fatty acid
initial concentration
and time of perfusion
Zero
(control 1)
0.075 m
M
palmitate
(control 2) 0.5 m
M
palmitate
20 min 60 min 60 min 20 min 60 min
AMP 0.131 ± 0.033 (5) 0.094 ± 0.011 (6) 0.092 ± 0.005 (7) 0.109 ± 0.013 (6) 0.095 ± 0.004 (7)
ADP 0.644 ± 0.048 (5) 0.478 ± 0.088 (6) 0.462 ± 0.021 (7) 0.577 ± 0.046 (6) 0.452 ± 0.014 (7)
ATP 2.19 ± 0.196 (5) 1.48 ± 0.294 (6) 2.09 ± 0.18 (7) 1.76 ± 0.233 (6) 1.84 ± 0.132 (7)
AMP/ATP ratio 0.063 ± 0.018 0.071 ± 0.011 0.047 ± 0.006 0.065 ± 0.009 0.054 ± 0.005
Energy charge 0.846 ± 0.018 0.844 ± 0.008 0.872 ± 0.009
a
0.833 ± 0.014 0.863 ± 0.010
Creatine ND 4.50 ± 0.66 (6) ND ND 3.70 ± 0.71 (6)
Phosphocreatine ND 6.54 ± 1.12 (6) ND ND 6.75 ± 1.08 (6)
Malonyl-CoA 3.35 ± 0.53 (6) 2.81 ± 0.33 (9) ND 2.77 ± 0.34 (6) 1.38 ± 0.13
b

M
palmitate
was seen when the perfusion time was 20 min (Fig. 4).
From this finding it was correctly predicted that 0.5 m
M
palmitate would have no significant effect at 20 min on the
content of the downstream marker malonyl-CoA (Table 3).
The emergence of a significant effect of palmitate between
20 and 60 min was not accompanied by any significant
changes in the AMP/ATP ratio or in the Ôenergy chargeÕ
(Table 3), providing further evidence that covalent activa-
tion of AMPKs following exposure to fatty acid was not
driven by changes in adenine nucleotides.
Cross-talk between the activation of AMPK by fatty acids
and insulin and adrenergic signalling processes
Fig. 5 shows studies focused on the dominant a-2 AMPK
isoform. Insulin decreased a-2 AMPK activity by 55% in
the absence of palmitate. This effect was prevented by
0.5 m
M
palmitate (Fig. 5A). As a consequence the 4-fold
increase due to palmitate in this series of experiments
became 10-fold when insulin was also present. Insulin also
significantly decreased a-1 AMPK activity by 81%
(P<0.05) from 2.74 ± 0.68–0.53 ± 0.31 pmolÆmin
)1
per mg protein—an effect that also was prevented by
0.5 m
M
palmitate (data not shown). As expected from

palmitate (filled symbols). AMPK
activity (expressed as pmolÆmin
)1
per mg 13 000 g supernatant pro-
tein) was measured without (squares) or with (circles) 200 l
M
AMP.
Values are means ± S.E.M. of 6–12 independent measurements. a,b,
indicate P < 0.01, < 0.001 for effects of palmitate vs. the control (at
60 min); c,d, indicate P < 0.05, P < 0.01 for comparison of 60 min
with 20 min values.
Fig. 5. Effects of palmitate, insulin and epinephrine on a-2 AMPK
activity and malonyl-CoA content. Hearts were perfused for 60 min
with 5 m
M
glucose and BSA (20 mgÆmL
)1
) and other additions as
indicated. C, No additions (control 1 conditions); I, 10 n
M
insulin,
E, 5 l
M
epinephrine; P, 0.5 m
M
palmitate; P + I, palmitate + insulin;
P + E, palmitate + epinephrine. The bars indicate ± S.E.M. Values
are means of between five and nine independent measurements. Open
bars: AMPK activity which was measured with 200 l
M

)1
pergwetweightofheartprovi-
ding reassurance that epinephrine was actually active under
these conditions.
Effect of fasting
in vivo
on AMPK activity
and the phosphorylation status of AMPK a-subunits
Fig. 6 shows that starvation for 24 h, which increased
serum NEFA concentration by almost 3-fold (and also
decreased blood glucose), significantly increased heart
P-T172 abundance by 2.2-fold and increased a-2 AMPK
activity to a similar extent. The a-2 AMPK activities in
Fig. 6 were appreciably lower than in Table 1 and in Figs 2,
4 and 5. In part this difference was due to the presence of
blood in these in vivo samples, i.e. the average protein in
13 000 g supernatantsfrom1gwetweightofperfused
heart was 58 mg whereas it was 130 mg for hearts sampled
in vivo (starvation had no effect on the protein content).
Also the goat antiserum used to immunoprecipitate the
AMPK for Fig. 6 yielded AMPK activities which were only
approximately half of those precipitated by the sheep
antiserum in all other experiments. Although these fed/
starved measurements were closely time-matched with each
other they were made some time after all of the perfusion
experiments. It is therefore possible that some degree of
animal variation could also have contributed to these
discrepancies.
Discussion
Our main conclusion was that an increase in NEFA

comparable to the highest similarly made measurements
that we could find in the literature [39,42,44,45]. Third, a
plot of the reciprocal of the increase in fatty acid utilization
by the hearts (Table 2) vs. 1/[NEFA] was linear (r ¼ 0.977,
P < 0.05) with half-maximal increase in fatty acid utiliza-
tion at 0.26 m
M
palmitate (NEFA/albumin ratio ¼
0.85 : 1) and V
max
for total fatty acid utilization at
17.2 lmolÆh
)1
per g wet weight or 75 lmolÆh
)1
per g dry
weight. This value is close to those of Saddik and
Lopaschuk [34] who perfused working rat hearts at a
NEFA/albumin ratio of 2.7 : 1 and observed rates of fatty
acid utilization of between 63 and 59 lmolÆh
)1
per g dry
weight through an initial ÔpulseÕ and subsequent ÔchaseÕ
period.
TheextentofactivationoftheAMPKwith0.5m
M
palmitate depended to some extent on the chosen control
Fig. 6. Effect of starvation on a-2 AMPK activity and on the phos-
phorylation state of Thr172 in AMPK a-subunits. Hearts were obtained
from fed (F) or 24 h-starved rats (S). Each of the measurements was

AMP/ATP ratio undetectable by present methods and it is
of note that some a-2 AMPK activity in heart is tightly
associated with ACC [19]. If we had found that no fatty
acids other than palmitate caused activation of the AMPK
it would have been reasonable to propose that sphingolipid
signalling processes might be involved because palmitate is a
metabolic precursor for sphingolipid signalling molecules
which in turn produce effects that are not seen with other
long-chain fatty acids [50–52]. However oleate was as
effective as palmitate in causing activation of AMPK
making an involvement of sphingolipid signalling unlikely.
Phosphorylation of Thr172 within AMPK a-subunits was a
significant feature of the activation of AMPK by fatty acids
andonethatiscommontotheÔclassicalÕ activation
pathway. However at this time we cannot discount the
possibility that exposure to fatty acids may promote other
phosphorylation events (e.g. within AMPK b-subunits)
which modify activity, subcellular localization or substrate
recognition [53–57]. It was noted that a-1, but not a-2
AMPK complexes lost sensitivity to AMP after exposure of
hearts to fatty acid (Table 1). The AMP binding site on
AMPK appears to be a higher order structure contributed
by two or more of the a, b and c subunits of the AMPK
heterotrimer [58,59]. It is possible that protein phosphory-
lation driven by NEFA selectively modifies this AMP
binding site in a-1 AMPK complexes.
Alone, epinephrine and palmitate each decreased malo-
nyl-CoA content (Fig. 5). Cyclic AMP-dependent protein
kinase
3

known. A novel and physiologically interesting observation
of the present study was that palmitate totally blocked
inactivation of the AMPK by insulin (Fig. 7), suggesting a
dominance of the fatty acid-driven pathway for activation
of AMPK over at least some aspects of insulin signalling.
This dominance of the fatty acid effect on AMPK activity
provides an explanation for the previous finding that
palmitate overrode the effect of insulin to increase malo-
nyl-CoA content in the heart [15,24]. It could also explain
why Sakamoto et al. [66] observed no effect of insulin on
heart AMPK activity since these authors perfused hearts
with 3% BSA and 0.4 m
M
or 1.2 m
M
palmitate. The study
of Gamble and Lopaschuk [65] though is at variance with
that of Sakamoto et al. [66]: Gamble and Lopaschuk [65]
used an identical perfusion system to Sakamoto et al. [66]
but reported that insulin caused a 40% decrease in AMPK
activity in hearts perfused with 3% BSA and 0.4 m
M
palmitate. However we have calculated that utilization of
fatty acid in Gamble and Lopaschuk’s experiments [65] was
approximately twice that reported by Sakamoto et al. [66].
The volume of the recirculated perfusate was not stated by
the former [65] and it is possible that their perfusate fatty
Fig. 7. Summary of the interplay between the effects of long-chain fatty
acid, insulin and epinephrine on AMPK activity and subsequent down-
stream changes to ACC, CPT1 and b-oxidation.

utilization of carbohydrate fuels when provision of NEFA
is increased [69]. A key feature of the Randle model is the
necessity for b-oxidation to increase prior to suppression
of carbohydrate utilization [70]. It is difficult to see how
this could be achieved without a preceding decrease in
malonyl-CoA sufficient to allow activation of CPT1, in
which case an early decrease in ACC activity (and/or an
increase in MCD activity) is also required. We have now
shown that this can be driven by an unidentified signalling
pathway though which increased NEFA activates the
AMPK. Carling et al. [71] reported that long-chain fatty
acyl-CoA can stimulate the phosphorylation and activa-
tion of AMPK in a semipurified system. However we have
no evidence for such a role of fatty acyl-CoA because in
cardiac myocytes 0.5 m
M
palmitate causes activation and
phosphorylation of AMPK to the same extent as in
perfused heart (Y. Tsuchiya and D. Saggerson, unpub-
lished data) without any change in the myocyte content of
fatty acyl-CoA [15].
Our finding that AMPK and malonyl-CoA are not
significantly changed after 20 min of perfusion (Fig. 4) is
potentially problematic. It could mean that these changes
are quite slow in onset, in which case they would not be
relevant to an acute ÔkickstartingÕ of the Randle cycle.
However removal from the anaesthetized animal followed
by cooling, cannulation and then the initiation of perfusion
will cause considerable metabolic stress to the heart which
could mask other underlying metabolic changes. The period

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