Covalent activation of heart AMP-activated protein kinase in responseto physiological concentrations of long-chain fatty acids Hilary Clark1, David Carling2and David Saggerson1 1 Departm
Trang 1Covalent activation of heart AMP-activated protein kinase in response
to physiological concentrations of long-chain fatty acids
Hilary Clark1, David Carling2and David Saggerson1
1
Department of Biochemistry and Molecular Biology, University College London, UK;2Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK
Rat hearts were perfused for 1 h with 5 mMglucose 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 inhibiactiva-tion of an AMPK
phos-phatase I n vivo, 24 h of starvation also increased heart AMPK activity and Thr172 phosphorylation of AMPK a-subunits Perfusion with insulin decreased both a-1 and a-2 AMPK activities and increased malonyl-CoA content Palmitate prevented both of these effects Perfusion with epinephrine decreased malonyl-CoA content without an effect on AMPK activity but prevented the activation of AMPK by palmitate The concept is discussed that activa-tion of AMPK by an unknown fatty acid-driven signalling process provides a mechanism for a feed-forward activation
of fatty acid oxidation
Keywords: AMP-activated protein kinase; fatty acids; heart; insulin; protein phosphorylation
The AMP-activated protein kinase (AMPK) is a
heterotri-meric enzyme complex with a key role in the regulation of
metabolism and other processes [1–4] AMPK is activated
following an increase in the cellular AMP/ATP ratio
Activation requires phosphorylation of Thr172 within the
a-subunit of AMPK, catalysed by an upstream AMPK
kinase Dephosphorylation of Thr172 (in vivo by
phospho-protein phosphatase 2C [5]) leads to inactivation of AMPK
Direct allosteric activation of AMPK also occurs following
an increase in the cellular AMP/ATP or
creatine/phospho-creatine ratios [6] These processes constitute the classical
pathway allowing AMPK to be a sensor of the cellular
energy charge under conditions of increased ATP
con-sumption and/or impeded ATP production Recently other
conditions have been described in which the AMPK is
covalently activated or inactivated without detectable
change in the cellular AMP/ATP ratio, e.g changes due
to insulin [7], leptin [8], metformin [9,10], hyperosmotic stress [9] and glucose deprivation [11] leading to proposals [9,11] that covalent activation of the AMPK may also occur through upstream processes independent of the classical 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 and an increased rate of b-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
Correspondence to D Saggerson, Department of Biochemistry and
Molecular Biology, University College London, Gower Street,
London, WC1E 6BT, UK.
Fax: + 44 20 7679 7193, Tel.: + 44 20 7679 7320,
E-mail: saggerson@biochem.ucl.ac.uk
Abbreviations: ACC, acetyl-CoA carboxylase; AMPK,
AMP-activa-ted protein kinase; CPT1, the overt form of mitochondrial carnitine
acyltransferase; KHB, Krebs–Henseleit bicarbonate; MCD,
malonyl-CoA decarboxylase; NEFA, non-esterified fatty acid; PKA, cyclic
AMP-dependent protein kinase; PP2C, phosphoprotein phosphatase
2C; PT-172, phosphorylation of Thr172 in AMPK a-subunits.
(Received 16 January 2004, revised 4 March 2004,
accepted 6 April 2004)
Trang 2phosphorylation mechanism that is not dependent upon
changes in the cellular AMP/ATP ratio Similar changes in
AMPK phosphorylation and activation were seen when rats
were starved for 24 h We also report on cross-talk between
this fatty acid-driven activation process and insulin and
adrenergic signalling pathways
Experimental procedures
Chemicals
Antisera against AMPK a-subunits were raised in sheep
[25] In one experiment (Fig 6) a goat antiserum (Santa
Cruz) was used These antisera or nonimmune serum were
prebound to protein G-Sepharose 4B Antibody against a
peptide surrounding phospho-Thr172 on the a-subunits of
AMPK was from New England Biolabs Antibody against
the phosphopeptide corresponding to amino acids 73–85 of
rat ACC-1 [HMRSSMS(PO4)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 20–22C 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 37C with
100 mL Krebs–Henseleit bicarbonate (KHB) medium
equilibrated with O2/CO2(19 : 1) containing 1.3 mMCaCl2,
5 mMglucose and fatty acid-poor BSA (20 mgÆmL)1) The
medium was recirculated except in experiments with
epinephrine The system [27] perfused the coronary
circu-lation and, because the ventricle was filled, required the
heart to work against a pressure of 80 ± 5 cm water Any
hearts which did not have a sustained and steady beat
throughout the experiment or which showed discoloured
regions denoting inadequate perfusion were discarded
After 20 or 60 min hearts were freeze-clamped (liquid
nitrogen) For in vivo measurements hearts from fed or
24 h-starved rats were directly freeze-clamped after removal
from the animal Procedures conformed to the UK Animals
(Scientific Procedures) Act, 1986
AMPK activity
Hearts were powdered under liquid nitrogen and
homo-genized (100 mgÆmL)1) in
homogenization/immunopreci-pitation buffer consisting of 50 mM Tris/HCl pH 7.8,
0.25 mM mannitol, 1 mM EDTA, 1 mM EGTA, 50 mM
NaF, 5 mM Na4P2O7, 1 mMdithiothreitol, 1 mM
phenyl-methanesulfonyl fluoride, 1 mMbenzamidine and soybean
trypsin inhibitor (4 lgÆmL)1) The homogenate was
centri-fuged at 4C for 10 min at 13 000 g and 250 lL of the
supernatant incubated for 2 h at 4C 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 (4C) with 300 lL of AMPK assay buffer (40 mM Hepes pH 7.0 contain-ing 80 mM NaCl, 0.8 mM EDTA, 8% v/v glycerol and
1 mM dithiothreitol) Finally washed immunoprecipitates were resuspended in 75 lL of AMPK assay buffer which additionally contained 200 lM SAMS peptide (HMRSAMSGLHLVKRR) [28], 5 mM MgCl2, with or without 200 lM AMP The AMPK assay was started by addition of 200 lM [c-33P]ATP (250–500 d.p.m.Æpmol)1) After 30 min at 37C the reaction was stopped by spotting
20 lL samples onto P81 Whatman phosphocellulose papers which were washed twice for 10 min in a solution of 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 (4C) with 300 lL
50 mM Tris/HCl pH 7.4 containing 1 mM dithiothreitol After recovery by centrifugation
immu-noprecipitates were resuspended in 25 lL of 50 mM Tris/ HCl pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, glycerol (5%, v/v) and PP2C (160 lgÆmL)1) MgCl2 was omitted from control incubations After 30 min at 30C the immunoprecipitates were again recovered by centrifugation and washed three times (4C) in 300 lL of AMPK assay buffer before resuspension in 75 lL of the same buffer together with the other components of the AMPK assay (see above) AMPK activity is expressed as pmolÆmin)1per
mg 13 000 g supernatant protein (i.e relative to the extract immediately before immunoprecipitation) Preliminary experiments established the optimum amounts of each anti-AMPK serum Blank activity with nonimmune sheep serum was subtracted
Western blotting Hearts were powdered under liquid nitrogen and homo-genized (200 mgÆmL)1) in homogenization/immunopreci-pitation buffer followed by centrifugation (13 000 g) for
5 min Supernatants (200 lg protein) were analysed by SDS/PAGE, transferred to poly(vinylidene difluoride) membranes and blotted with antiphospho-ACC Ig or antiphospho-AMPK primary Ig Following measurement
of phosphorylation of Thr172 within AMPK a-subunits (P-T172) blots were stripped to measure total abundance of AMPK a-subunits The membranes were left at 50C for
30 min in 62.5 mM Tris/HCl pH 6.8 containing 100 mM 2-mercaptoethanol and 2% SDS followed by three washes in
20 mMTris/HCl pH 7.5 containing 0.14MNaCl 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
Trang 3described [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 mMglucose alone or by
glucose with 0.075 mM, 0.25 mMor 0.5 mMlong-chain fatty
acid Palmitate (0.075 mM) was used because, with 2% BSA
present, this gave a NEFA/albumin molar ratio of
0.25 : 1, similar to that in fed plasma Palmitate/oleate
at 0.25/0.5 mM gave NEFA/albumin ratios representative
of mild (1 day) and severe (> 1 day) starvation,
respect-ively Over 60 min of perfusion we tested the effects of 0.25
and 0.5 mM fatty acids against two control conditions
(Table 1) Control 1 was where the initial perfusate fatty
acid concentration was zero This is technically the correct control but is unreal because a plasma NEFA concentration
of zero will not occur physiologically In fact hearts started with zero NEFA released a small but significant amount of NEFA over 60 min (Table 2) Control 2 was where hearts were started with 0.075 mMpalmitate Under this condition the hearts were essentially in NEFA balance whereas with 0.25 and 0.5 mMpalmitate net removal of NEFA from the perfusate occurred (Table 2)
Unexpectedly we found that a-1 and a-2 AMPK activities tended to be lowest in hearts started with 0.075 mM 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 mM palmitate increased a-1 and a-2 AMPK activities by at least 2-fold when AMP was omitted from these assays 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
in the perfusate
AMPK activity (pmolÆmin)1per mg of 13 000 g supernatant protein)
Assayed without
200 lm AMP
Assayed with AMP
Assayed without
200 lm AMP
Assayed with AMP
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 respectively versus 0.075 m M palmitate (unpaired test) i,j,k,l P < 0.02, < 0.025, < 0.01, < 0.0005 respectively for the effect of AMP (paired test).
Table 2 Net output or uptake of NEFA and glycerol by perfused hearts Hearts were perfused for 60 min with 5 m M glucose, BSA (20 mgÆml)1) and sodium palmitate as indicated Values are means ± S.E.M of between four and seven independent measurements.
Initial NEFA
concentration
in the perfusate
Final NEFA concentration in the perfusate (m M )
Change in perfusate NEFA (lmolÆh)1Æg wet wtÆheart)1) [A]
Glycerol release to perfusate (lmol of fatty acid equivalentÆ
h)1Æg wet wtÆheart)1) [B]
Total fatty acid utilisation (lmolÆh)1Æg wet wtÆheart)1) [B–A]
0.075 m M Palmitate
(control 2)
Trang 4a-2 AMPK these appeared to be two independent effects
(Table 1)
Using control 2 as the baseline, perfusion with 0.25 or
0.5 mMpalmitate increased a-1 AMPK activity by at least
3-fold and a-2 AMPK activity was increased by at least
2.5-fold by 0.25 mM palmitate and by approximately 3.5-fold
by 0.5 mMpalmitate These effects of palmitate were seen
with or without AMP in the assays (Table 1) Perfusion
with 0.5 mMoleate caused activation of both AMPKs to
levels similar to those seen with 0.5 mMpalmitate (Table 1)
Therefore the activation of AMPK was not peculiar to
palmitate but was a more generalized effect of long-chain
fatty acids
Downstream changes in ACC and malonyl-CoA
follow-ing activation of the AMPK were seen after perfusion with
0.5 mM palmitate for 60 min Phosphorylation of ACC
under control 1 conditions was virtually undetectable using
the antibody which recognizes the AMPK phosphorylation
site sequence SMS(PO4)GLHLVK in ACC-1 (265 kDa)
and also recognizes the equivalent AMPK phosphorylation
site in ACC-2 (280 kDa) However after perfusion with
0.5 mM 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 mMpalmitate (Fig 2) If Mg2+, 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 mM 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
Activation of AMPK by fatty acids does not require changes in cellular adenine nucleotides
Perfusion with 0.5 mM palmitate for 60 min had no significant effect on the contents of adenine nucleotides, creatine and phosphocreatine or on the AMP/ATP ratio and the energy charge [35] compared with either control 1
or control 2 conditions (Table 3) The only significant change that was seen was a very small increase in the energy
Fig 1 Effect of palmitate on the phosphorylation state of acetyl-CoA
carboxylase (Phospho-ACC) Hearts were perfused for 60 min with
5 m M glucose and BSA (20 mgÆmL)1) C, Zero NEFA (control 1
conditions); P, 0.5 m M palmitate Each of the measurements was
obtained from a separate heart.
Table 3 Measurements of adenine nucleotides, creatine, phosphocreatine and malonyl-CoA in perfused hearts Hearts were perfused for 20 or 60 min with 5 m M glucose, BSA (20 mgÆmL)1) and sodium palmitate as indicated Values are means ± S.E.M of the numbers of independent meas-urements shown in parentheses and are expressed as lmolÆg wet weight heart)1except for malonyl-CoA (nmolÆg wet weight)1) The energy charge 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
a,b P < 0.05, < 0.001, respectively, vs zero NEFA (unpaired test) ND, Not determined.
Fig 2 Effect of PP2C to abolish the activation of AMPK by palmitate Hearts were perfused for 60 min with 5 m M glucose and BSA (20 mgÆmL)1) without (open bars) or with 0.5 m M palmitate (filled bars) AMPK immunoprecipitates were incubated with 10 m M MgCl 2
and PP2C as indicated Incubation without MgCl 2 abolished effects of PP2C (data not shown) Values are means ± S.E.M of four inde-pendent measurements AMPK activity was measured without AMP and is expressed as pmolÆmin)1per mg 13 000 g supernatant protein.
Trang 5charge in control 2 compared with that in control 1
(Table 3)
Time-dependence of the activation of AMPK
by fatty acids
No significant activation of AMPKs by 0.5 mMpalmitate
was seen when the perfusion time was 20 min (Fig 4)
From this finding it was correctly predicted that 0.5 mM 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 energy charge (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 mM 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 mM palmitate (data not shown) As expected from previous studies [15,24] the content of the downstream marker malonyl-CoA altered inversely with these changes in AMPK activity (Fig 5A)
Fig 5B shows effects of epinephrine Epinephrine increased the rate of cardiac lipolysis measured as glycerol accumulation in the perfusate from 0.13 ± 0.03 to
Fig 3 Effect of palmitate on the phosphorylation state of Thr172 in
AMPK a-subunits Hearts were perfused for 60 min with 5 m M glucose
and BSA (20 mgÆmL)1) C, Zero NEFA (control 1 conditions);
P, 0.5 m M palmitate Each of the measurements was obtained from a
separate heart Band intensities from immunoblots were determined by
phosphoimaging These were expressed relative to the mean of the
values from hearts exposed to palmitate which was given an arbitrary
value of 1.0 (A) Means ± S.E.M for seven independent
measure-ments in both cases a, indicates P < 0.02 for the effect of palmitate.
(B) Representative images from immunoblots.
Fig 4 Time-dependence of activation of AMPK by palmitate Hearts
were perfused with 5 m M glucose and BSA (20 mgÆmL)1) without
(open symbols) or with 0.5 m M palmitate (filled symbols) AMPK
activity (expressed as pmolÆmin)1per 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 AMP present and is expressed as pmolÆmin)1per mg 13 000 g supernatant protein Filled bars: malonyl-CoA content expressed as nmol per g wet weight
of heart a,b,d, indicate P < 0.05, < 0.01, < 0.001 vs the control ( C); f,g, indicate P < 0.01vs insulin or vs epinephrine, respectively.
Trang 60.81 ± 0.16 lmolÆmin)1 per g wet weight of heart
(P < 0.01) For this reason the perfusate was not
recircu-lated in order to minimize any possible increase in AMPK
activity secondary to an increase in perfusate NEFA As
expected from previous studies [24,36] epinephrine alone
significantly decreased malonyl-CoA content However this
was not accompanied by any decrease in a-2 AMPK
activity Epinephrine also had no effect on a-1 AMPK
activity (data not shown) With epinephrine in combination
with 0.5 mMpalmitate, whilst the malonyl-coA content was
significantly lower than in the control condition, no
additivity in their effects on this parameter were seen
Furthermore, with epinephrine in combination with
palmi-tate, a-2 AMPK activity was not different from that in the
control condition, i.e epinephrine prevented the activating
effect of palmitate With epinephrine in combination with
palmitate the rate of glycerol release into the perfusate was
0.93 ± 0.23 lmolÆmin)1per g wet weight of heart
provi-ding reassurance that epinephrine was actually active under
these conditions
Effect of fastingin 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 supernatants from 1 g wet weight of perfused 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 characteristic of the fed to fasted transition led to phos-phorylation and activation of AMPK in perfused hearts without changes in contents of AMP, ATP, phosphocrea-tine and creaphosphocrea-tine Therefore consideration must be given to the likelihood of novel signalling processes that transmit, through a protein phosphorylation mechanism, information about the fat fuel availability or the relative fat/carbo-hydrate availability We are unaware of any previous report
of a such an effect of fatty acid in vitro except that Kawaguchi et al [37] showed that culture of hepatocytes with palmitate for 12 h increased AMPK activity—but with
a 30-fold increase in the AMP/ATP ratio We also showed that an increase in serum NEFA after 24 h of starvation was accompanied by increased AMPK activity and P-T172 abundance in vivo This increase in AMPK activity is likely
to be a contributing factor to the 70% decrease in heart malonyl-CoA after 24 h of starvation [38] although a decrease in insulin would also have some effect
We are satisfied that our heart perfusion conditions were adequately physiological and closely matched those in the literature as judged by three criteria First, 31 individual hearts gave values for the energy charge from 0.78 to 0.90 (Table 3) This range matches the highest values that we could find in the literature for similarly made measurements
in rat hearts perfused in the Langendorff mode with glucose
in KHB-based medium [7,39,40] and also matches values for hearts freeze-clamped in vivo [41,42] Second, using the estimate that the heart is 77% water (w/w) [43] to interconvert values expressed per g wet weight and per g dry weight our values for phosphocreatine (Table 3) were 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 (r¼ 0.977,
P< 0.05) with half-maximal increase in fatty acid utiliza-tion at 0.26 mM palmitate (NEFA/albumin ratio¼ 0.85 : 1) and Vmax for total fatty acid utilization at 17.2 lmolÆh)1per g wet weight or 75 lmolÆh)1per 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)1per g dry weight through an initial pulse and subsequent chase period
The extent of activation of the AMPK with 0.5 mM 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
obtained from a separate heart Band intensities from immunoblots
were determined by phosphoimaging These were expressed relative to
the mean of the values from hearts from starved rats which was given
an arbitrary value of 1.0 (A) Means ± S.E.M for measurements of
blood glucose and serum NEFA (n ¼ 6 for fed and 10 for starved,
respectively), a-2 AMPK activity assayed with and without AMP
(n ¼ 5–6) and P-T172 abundance (n ¼ 8) Open bars, fed; filled bars,
starved a,b,c,d, indicate P < 0.05, < 0.02, < 0.01, < 0.001,
respectively, for effects of starvation (B) Representative images from
immunoblots.
Trang 7conditions, the presence of AMP and whether or not insulin
was present In general, whatever the assay conditions, the
degree of activation was not trivial and was comparable
in scale with the activation of cardiac AMPK following
ischaemia [23], ischaemia/reperfusion [46,47] or anoxia [7]
Cardiac lipolysis is increased during ishaemia [48,49]
suggesting that part of the ischaemic increase in AMPK
activity could be secondary to provision of NEFA We also
suggest that increased AMPK activity in rat liver, adipose
tissue and skeletal muscle after treadmill running [22] may
be secondary to increased plasma NEFA
At present the signalling process through which increased
NEFA causes covalent activation of AMPK is unclear but
fatty acids must now join the list of agents which do this
without any of the relatively large changes in cellular
adenine nucleotides that are typical of the classical pathway
for AMPK activation However we cannot discount the
possibility that a subpopulation of AMPK and its upstream
kinase(s) are activated by a very localized change in the
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
and one that is common to the classical 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 (PKA) phosphorylates and inactivates heart ACC-2
[18,19,60] and isoprenaline increases phosphorylation of
ACC in cardiac myocytes [18] As epinephrine had no effect
on AMPK activity in the absence of palmitate it is likely
that it decreased malonyl-CoA through this
PKA-depend-ent mechanism The finding that epinephrine totally blocked
the activation of AMPK by palmitate is of note and requires
further investigation of the adrenergic signalling mechanism
that is involved Although effects of epinephrine and
palmitate to decrease malonyl-CoA were not additive, the
content of malonyl-CoA was still low when epinephrine and
palmitate were both present (Fig 5) suggesting that, whilst
phosphorylation/inactivation of ACC by AMPK is blocked
under these conditions, phosphorylation/inactivation of
ACC by PKA is still possible (summarized in Fig 7)
Phosphorylation of the rat ACC1 isoform on Ser77 by PKA blocks phosphorylation of Ser79 by AMPK and vice-versa [61], i.e phosphorylations of these two adjacent sites (which are also found in ACC2 [62]) are mutually exclusive Our findings now extend this notion of mutual exclusivity In the physiological context it is of note that neither epinephrine [63] nor cyclic AMP [64] enhanced cardiac oxidation of readily available fatty acid when carbohydrate was also available; rather, enhancement of carbohydrate usage was favoured
Insulin decreases heart AMPK activity under normal and anoxic conditions [7,65] and decreases the phosphorylation state of Thr172 within AMPK a-subunits [7] Apart from being blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin, no other details of this insulin process are 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 mMor 1.2 mMpalmitate 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 mM 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.
Trang 8acid had been depleted to such an extent that complete
blockade of insulin’s action was no longer seen
A covalent activation leading to
phosphorylation/inac-tivation of ACC, decreased malonyl-CoA and acphosphorylation/inac-tivation
of CPT1 provides a novel insight into ways in which a
feedforward activation of b-oxidation could occur in the
heart, and possibly in other cells/tissues Previously it has
been suggested for adipose tissue [67], skeletal muscle [68]
and hepatocytes [37] that AMPK could be activated
following increased AMP generation by increased flux of
fatty acid through fatty acyl-CoA synthetase However
our measurements of AMP and AMP/ATP do not
support this notion, at least in the heart From the data
given by Saddik and Lopaschuk [34] we have calculated
that activation of long-chain fatty acids to their CoA
thioesters by aerobic working rat heart accounted for only
0.6% of total ATP utilization when exogenous fatty acid
was absent This increased to only 1.9% of total ATP
utilization when exogenous fatty acid was high (1.2 mM
palmitate with 3% BSA) Therefore activation of
long-chain fatty acid is not an appreciable fraction of overall
cardiac ATP expenditure and is not likely to cause
appreciable perturbation of the AMP/ATP ratio Despite
our ignorance of the upstream mechanisms, a process for
feedforward activation of b-oxidation is of physiological
interest For example, it could provide a mechanism for
kickstarting Randle’s glucose fatty acid cycle which has
proposed an explanation for the acute decrease in
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 mM 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 kickstarting 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
of time that is necessary for the AMPK system to settle
down after this trauma is not known and requires
clarifi-cation by further experimental work Studies with rat
cardiac myocytes (Y Tsuchiya and D Saggerson,
unpub-lished data) have also shown that activation of AMPK by
palmitate is relatively slow
The list of the AMPK’s other immediate phosphorylation targets or downstream processes that are affected in various cells/tissues following activation of the AMPK is now very extensive and includes HMG-CoA reductase, mitochond-rial glycerolphosphate acyltransferase, nitric oxide synthase, hormone-sensitive lipase, creatine kinase, glycogen syn-thase, phosphofructokinase 2, ceramide synthesis, glucose uptake, apoptosis, insulin receptor substrate 1, mammalian target of rapamycin kinase (mTOR), mitogen-activated protein kinase kinase 3, c-Jun N-terminal kinase, translation elongation factor eEF2 and various transcriptional events (see Introduction for reviews) It is questionable whether it is desirable that all of these processes should be modified together following an elevation in plasma NEFA Therefore further work is needed to investigate the extent and time-scale over which NEFA-driven AMPK-mediated responses
in the heart extend beyond the ACC/MCD/malonyl-CoA/ CPT1 axis
Acknowledgements
This work was supported by the British Heart Foundation (H.C and D.S) and by the Medical Research Council (D.C).
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