Báo cáo khoa học: Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids docx

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 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 Ôfeed-forwardÕ 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 ÔclassicalÕ pathway allowing AMPK to be a sensor of the cellular Ôenergy chargeÕ 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 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 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) Eur. J. Biochem. 1–10 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04151.x phosphorylation 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 Ôcross-talkÕ 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(PO 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 glucose 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 m M Tris/HCl pH 7.8, 0.25 m M mannitol, 1 m M EDTA, 1 m M EGTA, 50 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- ing 80 m M NaCl, 0.8 m M EDTA, 8% v/v glycerol and 1m M dithiothreitol). Finally washed immunoprecipitates were resuspended in 75 lL of AMPK assay buffer which additionally contained 200 l M ÔSAMSÕ peptide (HMRSAMSGLHLVKRR) [28], 5 m M MgCl 2 ,withor without 200 l M AMP. The AMPK assay was started by addition of 200 l M [c- 33 P]ATP (250–500 d.p.m.Æpmol )1 ). After 30 min at 37 °C 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 (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 and washed three times (4 °C) 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 )1 per 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 50 °Cfor 30 min in 62.5 m M Tris/HCl pH 6.8 containing 100 m M 2-mercaptoethanol and 2% SDS followed by three washes in 20 m 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 ,0.25 m M or 0.5 m M long-chain fatty acid. Palmitate (0.075 m M ) 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 m M 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 m M 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 m M palmitate. Under this condition the hearts were essentially in ÔNEFA balanceÕ whereas with 0.25 and 0.5 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 in the perfusate AMPK activity (pmolÆmin )1 per mg of 13 000 g supernatant protein) a-1 Complexes a-2 Complexes Assayed without 200 lmAMP Assayed with AMP Assayed without 200 lmAMP Assayed with AMP Zero (control 1) 1.09 ± 0.27 (11) 2.70 ± 0.68 j (6) 2.11 ± 0.49 (11) 4.21 ± 0.35 l (11) 0.075 m M Palmitate (control 2) 0.70 ± 0.15 (5) 0.97 ± 0.30 a (5) 1.49 ± 0.30 (6) 2.27 ± 0.63 b,I (6) 0.25 m M Palmitate 2.46 ± 0.64 b,e 3.48 ± 0.92 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 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 )1 Æg wet wtÆheart )1 ) [A] Glycerol release to perfusate (lmol of fatty acid equivalentÆ h )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 0.5 m M palmitate 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 m M palmitate and by approximately 3.5-fold by 0.5 m M palmitate. These effects of palmitate were seen with or without AMP in the assays (Table 1). Perfusion with 0.5 m M oleate caused activation of both AMPKs to levels similar to those seen with 0.5 m M palmitate (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 m M 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(PO 4 )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 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. Activation of AMPK by fatty acids does not require changes in cellular adenine nucleotides Perfusion with 0.5 m M 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 Ôenergy chargeÕ [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 Ôenergy Fig. 1. Effect of palmitate on the phosphorylation state of acetyl-CoA carboxylase (Phospho-ACC). Hearts were perfused for 60 min with 5m 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. Heartswereperfusedfor20or60min 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 )1 except for malonyl-CoA (nmolÆgwetweight )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 (9) 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 )1 per mg 13 000 g supernatant protein. 4 H. Clark et al. (Eur. J. Biochem.) Ó FEBS 2004 chargeÕ 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 m 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 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 valuesfromheartsexposedtopalmitatewhichwasgivenanarbitrary value of 1.0. (A) Means ± S.E.M. for seven independent measure- mentsinbothcases.a,indicatesP < 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 )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 AMP present and is expressed as pmolÆmin )1 per 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,<0.001vs. thecontrol (C);f,g,indicateP < 0.01vs. insulin or vs. epinephrine, respectively. Ó FEBS 2004 Activation of AMPK by fatty acids (Eur. J. Biochem.)5 0.81 ± 0.16 lmolÆmin )1 pergwetweightofheart (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 m M palmitate, 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 )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 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 creatine. 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 Ôenergy chargeÕ 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 (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 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. 6 H. Clark et al. (Eur. J. Biochem.) Ó FEBS 2004 conditions, 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 ÔclassicalÕ 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 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 (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 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. Ó FEBS 2004 Activation of AMPK by fatty acids (Eur. J. Biochem.)7 acid 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 activation of CPT1 provides a novel insight into ways in which a ÔfeedforwardÕ 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 m M 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 ÔfeedforwardÕ activation of b-oxidation is of physiological interest. For example, it could provide a mechanism for ÔkickstartingÕ Randle’s Ôglucose fatty acid cycleÕ 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 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 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). References 1. Leclerc, I., Viollet, B., da Silva Xavier, G., Kahn, A. & Rutter, G.A. (2002) Role of AMP-activated protein kinase in the regulation of gene transcription. Biochem. Soc. Trans. 30, 307– 311. 2. Kemp, B.E., Stapleton, D., Campbell, D.J., Chen, Z.P., Murthy, S., Walter, M., Gupta, A., Adams, J.J., Katsis, F., van Denderen, B.,Jennings,I.G.,Iseli,T.,Michell,B.J.&Witters,L.A.(2003) AMP-activated protein kinase, super metabolic regulator. Biochem. Soc. Trans. 31, 162–168. 3. Hue, L., Beauloye, C., Bertrand, L., Horman, S., Krause, U., Marsin, A.S., Meisse, D., Vertommen, D. & Rider, M.H. (2003) New targets of AMP-activated protein kinase. Biochem. Soc. Trans. 31, 213–215. 4. Hardie, D.G. (2003) Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endo- crinology 144, 5179–5183. 5. Moore, F., Weekes, J. & Hardie, D.G. (1991) Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. Eur. J. Biochem. 199, 691–697. 6. Ponticos, M., Lu, Q.L., Morgan, J.E., Hardie, D.G., Partridge, T.A. & Carling, D. (1998) Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle. EMBO J. 17, 1688–1699. 7. Beauloye, C., Marsin, A.S., Bertrand, L., Krause, U., Hardie, D.G., Vanoverschelde, J.L. & Hue, L. (2001) Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett. 505, 348–352. 8. Minokoshi, Y., Kim, Y B., Peroni, O.D., Fryer, L.G.D., Muller, C., Carling, D. & Kahn, B.B. (2002) Leptin stimulates fatty acid oxidation by activating AMP-activated protein kinase. Nature (London) 415, 339–343. 9. Fryer, L.G., Parbu-Patel, A. & Carling, D. (2002) The Anti-dia- betic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J. Biol. Chem. 277, 25226–25232. 8 H. Clark et al. (Eur. J. Biochem.) Ó FEBS 2004 10. Hawley, S.A., Gadalla, A.E., Olsen, G.S. & Hardie, D.G. (2002) The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes 51, 2420–2425. 11. Itani, S.I., Saha, A.K., Kurowski, T.G., Coffin, H.R., Tornheim, K. & Ruderman, N.B. (2003) Glucose Autoregulates Its Uptake in Skeletal Muscle: Involvement of AMP-Activated Protein Kinase. Diabetes 52, 1635–1640. 12. Hong, S.P., Leiper, F.C., Woods, A., Carling, D. & Carlson, M. (2003) Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl Acad. Sci. USA 100, 8839–8843. 13. Hawley, S.A., Boudeau, J., Reid, J.L., Mustard, K.J., Udd, L., Makela, T.P., Alessi, D.R. & Hardie, D.G. (2003) Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J. Biol. 2, 28. 14. Dyck, J.R. & Lopaschuk, G.D. (2002) Malonyl CoA control of fatty acid oxidation in the ischemic heart. J. Mol. Cell. Cardiol. 34, 1099–1109. 15. Hamilton, C. & Saggerson, E.D. (2000) Malonyl-CoA metabolism in cardiac myocytes. Biochem. J. 350, 61–67. 16. Saggerson, E.D. & Carpenter, C.A. (1981) Carnitine palmitoyl- transferase and carnitine octanoyltransferase activities in liver, kidney cortex, adipocyte, lactating mammary gland, skeletal muscle and heart. FEBS Lett. 129, 229–232. 17. Brownsey, R.W., Zhande, R. & Boone, A.N. (1997) Isoforms of acetyl-CoA carboxylase: structures, regulatory properties and metabolic functions. Biochem. Soc. Trans. 25, 1232–1238. 18. Boone, A.N., Rodrigues, B. & Brownsey, R.W. (1999) Multiple- site phosphorylation of the 280 kDa isoform of acetyl-CoA car- boxylase in rat cardiac myocytes: evidence that cAMP-dependent protein kinase mediates effects of beta-adrenergic stimulation. Biochem. J. 341, 347–354. 19. Dyck, J.R., Kudo, N., Barr, A.J., Davies, S.P., Hardie, D.G. & Lopaschuk, G.D. (1999) Phosphorylation control of cardiac acetyl-CoA carboxylase by cAMP- dependent protein kinase and 5¢-AMP activated protein kinase. Eur. J. Biochem. 262, 184–190. 20. Saha, A.K., Schwarsin, A.J., Roduit, R., Masse, F., Kaushik, V., Tornheim, K., Prentki, M. & Ruderman, N.B. (2000) Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contrac- tion and the AMP-activated protein kinase activator 5- amino- imidazole-4-carboxamide-1-beta-D-ribofuranoside. J. Biol. Chem. 275, 24279–24283. 21. Habinowski, S.A., Hirshman, M., Sakamoto, K., Kemp, B.E., Gould, S.J., Goodyear, L.J. & Witters, L.A. (2001) Malonyl-CoA decarboxylase is not a substrate of AMP-activated protein kinase in rat fast-twitch skeletal muscle or an islet cell line. Arch. Biochem. Biophys. 396, 71–79. 22. Park, H., Kaushik, V.K., Constant, S., Prentki, M., Przybyt- kowski,E.,Ruderman,N.B.&Saha,A.K.(2002)Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phos- phate acyltransferase, and acetyl-CoA carboxylase by AMP-acti- vated protein kinase in rat tissues in response to exercise. J. Biol. Chem. 277, 32571–32577. 23. Kudo,N.,Barr,A.J.,Barr,R.L.,Desai,S.&Lopaschuk,G.D. (1995) High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5¢-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J. Biol. Chem. 270, 17513– 17520. 24. Awan, M.M. & Saggerson, E.D. (1993) Malonyl-CoA metabolism in cardiac myocytes and its relevance to the control of fatty acid oxidation. Biochem. J. 295, 61–66. 25. Woods, A., Salt, I., Scott, J., Hardie, D.G. & Carling, D. (1996) The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett. 397, 347–351. 26. Evans, W.H. & Mueller, P.S. (1963) Effects of palmitate on the metabolism of leukocytes from guinea pig exudate. J. Lipid. Res. 4, 39–45. 27. Mowbray, J. & Ottaway, J.H. (1973) The flux of pyruvate in perfused rat heart. Eur. J. Biochem. 36, 362–368. 28. Davies, S.P., Carling, D. & Hardie, D.G. (1989) Tissue distribu- tion of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur. J. Biochem. 186, 123–128. 29. Lawson, R. & Mowbray, J. (1986) Purine nucleotide metabolism: the discovery of a major new oligomeric adenosine tetraphosphate derivative in rat heart. Int.J.Biochem.18, 407–413. 30. Wahlefeld, A W. & Siedel, J. (1985) Creatine and Creatinine. In Methods of Enzymatic Analysis (Bergmeyer, H.U., Bergmeyer, J. & Grassl, M., eds), pp. 500–507. VCH Publishers, Deerfield Beach. 31. Heinz, F. & Weisser, H. (1985) Creatine Phosphate. In Methods of Enzymatic Analysis (Bergmeyer,H.U.,Bergmeyer,J.&Grassl, M., eds), pp. 507–514. VCH Publishers, Deerfield Beach. 32. Garland, P.B. & Randle, P.J. (1962) A rapid enzymic assay for glycerol. Nature (London) 196, 987–988. 33. Kunst, A., Draeger, B. & Ziegenhorn, J. (1985) D-Glucose. In Methods of Enzymatic Analysis (Bergmeyer, H.U., Bergmeyer, J. & Grassl, M., eds), pp. 163–172. VCH Publishers, Deerfield Beach. 34. Saddik, M. & Lopaschuk, G.D. (1991) Myocardial triglyceride turnover and contribution to energy substrate utilization in iso- lated working rat hearts. J. Biol. Chem. 266, 8162–8170. 35. Atkinson, D.E. (1977) Cellular Energy Metabolism and its Regu- lation. Academic Press, New York. 36. Goodwin, G.W., Taylor, C.S. & Taegtmeyer, H. (1998) Regula- tion of energy metabolism of the heart during acute increase in heart work. J. Biol. Chem. 273, 29530–29539. 37. Kawaguchi, T., Osatomi, K., Yamashita, H., Kabashima, T. & Uyeda, K. (2002) Mechanism for fatty acid ÔsparingÕ effect on glucose-induced transcription: regulation of carbohydrate- responsive element-binding protein by AMP-activated protein kinase. J. Biol. Chem. 277, 3829–3835. 38. McGarry, J.D., Mills, S.E., Long, C.S. & Foster, D.W. (1983) Observations on the affinity for carnitine, and malonyl-CoA sen- sitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non- hepatic tissues of the rat. Biochem. J. 214, 21–28. 39. Raatikainen, M.J., Peuhkurinen, K.J. & Hassinen, I.E. (1994) Contribution of endothelium and cardiomyocytes to hypoxia-induced adenosine release. J. Mol. Cell Cardiol. 26, 1069– 1080. 40. Khatib, S.Y., Farah, H. & El-Migdadi, F. (2001) Allopurinol enhances adenine nucleotide levels and improves myocardial function in isolated hypoxic rat heart. Biochemistry (Moscow) 66, 328–333. 41. Casey, T.M., Dufall, K.G. & Arthur, P.G. (1999) An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state. Eur. J. Biochem. 261, 740– 745. 42. Tveita, T., Skandfer, M., Refsum, H. & Ytrehus, K. (1996) Experimental hypothermia and rewarming: changes in mechanical function and metabolism of rat hearts. J. Appl. Physiol. 80, 291–297. 43. Diem, K. (1962) Composition of the body, body fluids and secretions. In: Documenta Geigy, Scientific Tables. (Diem, K., ed.), pp. 516–598. Geigy Pharmaceutical Co., Ltd., Manchester. 44. Hearse, D.J., Ferrari, R. & Sutherland, F.J. (1999) Cardiopro- tection: intermittent ventricular fibrillation and rapid pacing can Ó FEBS 2004 Activation of AMPK by fatty acids (Eur. J. Biochem.)9 induce preconditioning in the blood-perfused rat heart. J. Mol. Cell. Cardiol. 31, 1961–1973. 45. Smolenski, R.T., Amrani, M., Jayakumar, J., Jagodzinski, P., Gray, C.C., Goodwin, A.T., Sammut, I.A. & Yacoub, M.H. (2001) Pyruvate/dichoroacetate supply during reperfusion accel- erates recovery of cardiac energetics and improves mechanical function following cardioplegic arrest. Eur. J. Cardio-Thorac Surg. 19, 865–872. 46. Kudo, N., Gillespie, J.G., Kung, L., Witters, L.A., Schulz, R., Clanachan, A.S. & Lopaschuk, G.D. (1996) Characterization of 5¢AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim. Biophys. Acta. 1301, 67–75. 47. Lopaschuk, G.D. (1997) Alterations in fatty acid oxidation during reperfusion of the heart after myocardial ischemia. Am. J Cardiol. 80, 11A–16A. 48. Trach, V., Buschmans-Denkel, E. & Schaper, W. (1986) Relation between lipolysis and glycolysis during ischemia in the isolated rat heart. Basic Res. Cardiol. 81, 454–464. 49. van Bilsen, M., van der Vusse, G.J., Willemsen, P.H., Coumans, W.A., Roemen, T.H. & Reneman, R.S. (1989) Lipid alterations in isolated, working rat hearts during ischemia and reperfusion: its relation to myocardial damage. Circ. Res. 64, 304–314. 50. de Vries, J.E., Vork, M.M., Roemen, T.H., de Jong, Y.F., Cleutjens, J.P., van der Vusse, G.J. & van Bilsen, M. (1997) Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes. J. Lipid Res. 38, 1384–1394. 51. Hickson-Bick, D.L., Buja, M.L. & McMillin, J.B. (2000) Palmi- tate-mediated alterations in the fatty acid metabolism of rat neo- natal cardiac myocytes. J. Mol. Cell Cardiol. 32, 511–519. 52. Sparagna, G.C., Hickson-Bick, D.L., Buja, L.M. & McMillin, J.B. (2000) A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis. Am.J.Physiol.HeartCirc.Physiol. 279, H2124–H2132. 53. Hawley, S.A., Davison, M., Woods, A., Davies, S.P., Beri, R.K., Carling, D. & Hardie, D.G. (1996) Characterization of the AMP- activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP- activated protein kinase. J. Biol. Chem. 271, 27879–27887. 54. Stein,S.C.,Woods,A.,Jones,N.A.,Davison,M.D.&Carling,D. (2000) The regulation of AMP-activated protein kinase by phos- phorylation. Biochem. J. 345, 437–443. 55. Mitchelhill, K.I., Michell, B.J., House, C.M., Stapleton, D., Dyck, J.,Gamble,J.,Ullrich,C.,Witters,L.A.&Kemp,B.E.(1997) Posttranslational modifications of the 5¢-AMP-activated protein kinase beta1 subunit. J. Biol. Chem. 272, 24475–24479. 56. Warden, S.M., Richardson, C., O’Donnell, J. Jr, Stapleton, D., Kemp, B.E. & Witters, L.A. (2001) Post-translational modif- ications of the beta-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization. Biochem. J. 354, 275–283. 57. Woods,A.,Vertommen,D.,Neumann,D.,Turk,R.,Bayliss,J., Schlattner,U.,Wallimann,T.,Carling,D.&Rider,M.H.(2003) Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J. Biol. Chem. 278, 28434– 28442. 58. Kemp, B.E., Mitchelhill, K.I., Stapleton, D., Michell, B.J., Chen, Z.P. & Witters, L.A. (1999) Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem. Sci. 24, 22–25. 59. Cheung, P.C., Salt, I.P., Davies, S.P., Hardie, D.G. & Carling, D. (2000) Characterization of AMP-activated protein kinase gamma- subunit isoforms and their role in AMP binding. Biochem. J. 346, 659–669. 60.Winz,R.,Hess,D.,Aebersold,R.&Brownsey,R.W.(1994) Unique structural features and differential phosphorylation of the 280- kDa component (isozyme) of rat liver acetyl-CoA carbox- ylase. J. Biol. Chem. 269, 14438–14445. 61. Munday, M.R., Carling, D. & Hardie, D.G. (1988) Negative in- teractions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases. FEBS Lett. 235, 144–148. 62. Abu-Elheiga, L., Almarza-Ortega, D.B., Baldini, A. & Wakil, S.J. (1997) Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal mapping, and evidence for two isoforms. J. Biol. Chem. 272, 10669–10677. 63. Goodwin,G.W.,Ahmad,F.,Doenst,T.&Taegtmeyer,H.(1998) Energy provision from glycogen, glucose, and fatty acids on adrenergic stimulation of isolated working rat hearts. Am. J. Physiol. 274, H1239–H1247. 64. Depre,C.,Ponchaut,S.,Deprez,J.,Maisin,L.&Hue,L.(1998) Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart. J. Clin. Invest. 101, 390–397. 65. Gamble, J. & Lopaschuk, G.D. (1997) Insulin inhibition of 5¢-adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhi- bition of fatty acid oxidation. Metabolism 46, 1270–1274. 66. Sakamoto, J., Barr, R.L., Kavanagh, K.M. & Lopaschuk, G.D. (2000) Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart. Am. J. Physiol. Heart Circ. Physiol. 278, H1196–H1204. 67. Hardie, D.G. & Carling, D. (1997) The AMP-activated protein kinase – fuel gauge of the mammalian cell? Eur. J. Biochem. 246, 259–273. 68. Alam, N. & Saggerson, E.D. (1998) Malonyl-CoA and the reg- ulation of fatty acid oxidation in soleus muscle. Biochem. J. 334, 233–241. 69. Randle, P.J., Garland, P.B., Hales, C.N. & Newsholme, E.A. (1963) The glucose fatty acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet i, 785–789. 70. Caterson, I.D., Fuller, S.J. & Randle, P.J. (1982) Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid on pyruvate dehydrogenase complex activity in starved and alloxan- diabetic rats. Biochem. J. 208, 53–60. 71. Carling, D., Zammit, V.A. & Hardie, D.G. (1987) A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett. 223, 217–222. 10 H. Clark et al. (Eur. J. Biochem.) Ó FEBS 2004 . Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids Hilary Clark 1 ,. oxidation. Keywords: AMP-activated protein kinase; fatty acids; heart; insulin; protein phosphorylation. The AMP-activated protein kinase (AMPK) is a heterotri- meric