The Relationship between AMP-activated Protein Kinase Activity and AMP Concentration in the Isolated Perfused Rat Heart
2002; Elsevier BV; Volume: 277; Issue: 3 Linguagem: Inglês
10.1074/jbc.m107128200
ISSN1083-351X
AutoresMarkus Frederich, James A. Balschi,
Tópico(s)Cardiovascular Function and Risk Factors
ResumoThe objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of β-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by 31P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 μm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol·min−1·μg of protein−1. The half-maximal AMPK activation (A0.5) was 1.8 ± 0.3 μmAMP. Measurements in vitro have reported similar AMPKA0.5 at 0.2 mm ATP, but found thatA0.5 increased 10–20-fold at 4 mmATP. The low A0.5 of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart. The objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of β-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by 31P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 μm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol·min−1·μg of protein−1. The half-maximal AMPK activation (A0.5) was 1.8 ± 0.3 μmAMP. Measurements in vitro have reported similar AMPKA0.5 at 0.2 mm ATP, but found thatA0.5 increased 10–20-fold at 4 mmATP. The low A0.5 of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart. AMP-activated protein kinase acetyl-CoA-carboxylase bromo-octanoate amino-oxyacetate phosphocreatine intracellular pH Krebs-Henseleit buffer phenylphosphonic acid beats/min AMP-activated protein kinase (AMPK)1 and AMPK kinase comprise a protein kinase cascade that has been highly conserved throughout evolution (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar, 2Hardie D.G. Carling D. Carlson M. Ann. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1266) Google Scholar). Increases in AMP concentration ([AMP]) activate this cascade by four mechanisms (3Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 4Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (492) Google Scholar, 5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar). These mechanisms are as follows: 1) an allosteric activation by AMP of AMPK kinase, which then phosphorylates AMPK; 2) the binding of AMP to AMPK, which makes it a poorer substrate for protein phosphatases; 3) the binding of AMP to AMPK, which makes AMPK a better substrate for AMPK kinase; and 4) the allosteric activation by AMP of AMPK. The activating effects of AMP are antagonized by high concentrations of ATP. Since the AMPK is activated when AMP is elevated and ATP is depressed, AMPK is hypothesized to act as cellular "fuel gauge" (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar).After AMPK activation, in response to metabolic stress, AMPK phosphorylates enzymes, leading to activation of catabolic pathways to increase ATP synthesis and inhibition of anabolic pathways to limit ATP consumption. For example, AMPK phosphorylation decreases acetyl-CoA-carboxylase (ACC) activity, which decreases malonyl-CoA concentration. Malonyl-CoA inhibits carnitine palmitoyltransferase-1, which transports fatty acids into the mitochondrion (6Lopaschuk G.D. Am. J. Cardiol. 1997; 80: 11A-16AAbstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 7Muoio D.M. Seefeld K. Witters L.A. Coleman R.A. Biochem. J. 1999; 338: 783-791Crossref PubMed Scopus (342) Google Scholar). Reduction of malonyl-CoA increases fatty acid uptake via carnitine palmitoyltransferase-1 and, thereby, the fatty acid oxidation by the mitochondria, which increases ATP production. In skeletal muscle and hearts, AMPK activity also increases glucose uptake by enhancing GLUT-4 translocation (8Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Crossref PubMed Scopus (583) Google Scholar, 9Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (379) Google Scholar, 10Russell III, R.R. Bergeron R. Shulman G.I. Young L.H. Am. J. Physiol. 1999; 277: H643-H649PubMed Google Scholar). In hearts, AMPK phosphorylation of 6-phosphofructo-2-kinase increases fructose 2,6-bisphosphate, a stimulator of 6-phosphofructo-1-kinase, which accelerates glycolysis (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). AMPK also down-regulates ATP-consuming pathways, such as glycogen, cholesterol, and fatty acid synthesis (12Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar, 13Vavvas D. Apazidis A. Saha A.K. Gamble J. Patel A. Kemp B.E. Witters L.A. Ruderman N.B. J. Biol. Chem. 1997; 272: 13255-13261Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar).Several studies have examined the relationship between the activation of AMPK and [AMP]. Hardie et al. (14Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (315) Google Scholar) modeled the AMPK cascade. Their model predicts a sigmoidal response of AMPK activity to increasing [AMP] with a half-maximal activation (A0.5) of 4 μm. Stein et al. measured AMPK activity of the α1 and α2 isoforms in vitro as a function of [AMP] (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar). These measurements showed a hyperbolic increase in AMPK activity with increasing [AMP], with an A0.5 of 5.7 μm for α1 and 16 μm for α2. In contrast, Marsin et al. (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar) reported a linear relationship between AMPK activity and AMP/ATP ratio, using total AMP measured in tissue extracts of isolated rat hearts during ischemia or treatment with inhibitors of ATP synthesis.The aim of the present study was to define the relationship between AMPK activation and cytosolic [AMP] in vivo in the isolated perfused rat heart. To accomplish this, we employed an approach that restrains substrate metabolism by use of the inhibitors bromo-octanoate (BrO) to inhibit fatty acid oxidation (16Schulz H. Life Sci. 1987; 40: 1443-1449Crossref PubMed Scopus (52) Google Scholar) and amino-oxyacetate (AOA), to inhibit pyruvate oxidation (17Safer B. Smith C.M. Williamson J. J. Mol. Cell Cardiol. 1971; 2: 111-124Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Using this approach together with increased work demand reduced the phosphocreatine (PCr) (18Balschi J.A. Shen H. Madden M.C. Hai J.O. Bradley Jr., E.L. Wolkowicz P.E. J. Mol. Cell. Cardiol. 1997; 29: 3123-3133Abstract Full Text PDF PubMed Scopus (13) Google Scholar). We used 31P NMR spectroscopy to measure the PCr and ATP content as well as the intracellular pH (pHi) of these hearts. The creatine kinase and the adenylate kinase equilibrium expressions were used to calculate [ADP] and [AMP], respectively. The net effect of [PCr] reductions is an increase in [AMP] with a relatively constant [ATP]. Cytosolic [AMP] was therefore manipulated in a relatively controlled manner, and the AMPK activity was measured.DISCUSSIONResults in this paper define the relationship between AMPK activity and the cytosolic [AMP] in the isolated perfused rat heart. To our knowledge, this is the first correlation of AMPK activity with metabolically active cytosolic [AMP] in the isolated heart. To increase the cytosolic [AMP] in isolated perfused rat hearts, the heart's supply of acetyl-CoA was limited using the inhibitors bromo-octanoate and amino-oxyacetate. This method decreases both aerobic energy production and high energy phosphates, particularly PCr (18Balschi J.A. Shen H. Madden M.C. Hai J.O. Bradley Jr., E.L. Wolkowicz P.E. J. Mol. Cell. Cardiol. 1997; 29: 3123-3133Abstract Full Text PDF PubMed Scopus (13) Google Scholar). Because of the equilibrium of the creatine kinase reaction, the reduction of [PCr] results in an increase in [ADP]. In turn, the near equilibrium of the adenylate kinase reaction translates the increase in [ADP] into an increase in [AMP]. 31P NMR-measured PCr, ATP, and pHi provided an estimate of cytosolic [AMP] in vivo.Four conclusions can be drawn from the AMPK activity and [AMP] measurements of the seven groups of hearts studied here. First, Glc hearts, oxidizing both exogenous and endogenous glucose and endogenous triglycerides, maintain low values of [AMP] and AMPK activity. Second, AMPK activity in GBA-Bu hearts treated with inhibitors of fatty acid and glucose oxidation but provided with the substrate butyrate, which can bypass the blockade of β-oxidation, is equal to that of the Glc group hearts. These hearts maintain [AMP] equal to those of Glc hearts. These results show that AMPK activity does not increase due to nonspecific effects of the inhibitors. Third, AMPK activity increased in the GA hearts paced at 450 bpm. Fourth, GBA hearts treated with BrO and AOA to inhibit fatty acid and glucose oxidation have increased [AMP] as well as increased AMPK activity. In this study, hearts with an [AMP] above 10 μm demonstrate maximal AMPK activity (Fig. 2).Maximal increases in AMPK activity have been reported by a number of investigators. In vitro measurements by Stein found a 300% maximal activity for the α1 AMPK isoform and a 1300% maximal activity for the α2 AMPK isoform (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar). The heart contains both AMPK isoforms with the α2 isoform accounting for 70–80% of the total (27Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 28Dyck J.R. Kudo N. Barr A.J. Davies S.P. Hardie D.G. Lopaschuk G.D. Eur. J. Biochem. 1999; 262: 184-190Crossref PubMed Scopus (135) Google Scholar). Kudo et al. (29Kudo N. Barr A.J. Barr R.L. Desai S. Lopaschuk G.D. J. Biol. Chem. 1995; 270: 17513-17520Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar) reported a 200% increase in AMPK activity in the heart after 30 min of ischemia. A study by Marsinet al. (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar) of AMPK activity during ischemia found a nearly 800% increase after 10 min of ischemia, which decreased to a 500% increase after 30 min of ischemia. In the present study, the maximal increase in AMPK activity observed was ∼240% (relative to Glc, TableII).To investigate whether AMPK activity could be greater than the 240% that we observed in this study during ischemia, we measured AMPK activity (without AMP) after 30 min of ischemia. The AMPK activity, 107 pmol·min−1·μg of protein−1, is an increase of ∼500% higher than the Glc group. Thus, the AMPK activity after 30 min of ischemia was approximately twice the maximal activity observed (∼240%) in the groups of this study. The greater AMPK activity observed in the ischemic heart could result from activation due to higher [AMP]. During no-flow ischemia in the rat heart, [AMP] is higher for at least two reasons: first, [PCr] falls to nondetectable levels in about 10 min; second, pHi decreases to 6.3 within 10 min. Cytosolic AMP-specific 5′-nucleotidase, which deaminates AMP to adenosine and Pi, is inhibited at a pHi of 6.3 (30Bak M.I. Ingwall J.S. Am. J. Physiol. 1998; 274: C992-C1001Crossref PubMed Google Scholar). Inhibition of 5′-nucleotidase allows AMP to accumulate to 143 μm after 12 min of ischemia (30Bak M.I. Ingwall J.S. Am. J. Physiol. 1998; 274: C992-C1001Crossref PubMed Google Scholar). The conditions employed in the present study allow controlled increases in [AMP], but since 5′-nucleotidase is not inhibited, AMP does not accumulate. Hence, the AMPK activity achieved here may not be maximal.[AMP] and AMPK CascadeUsing our methods to alter myocardial energy metabolism, the cytosolic [AMP] ranged from 0.2 to 28 μm. The A0.5 = 1.8 μm AMP suggests that the threshold for [AMP] activation of AMPK in the heart is low and that AMPK activity is maximal at [AMP] ≥ 10 μm. In vitro the relationship between AMPK activation and [AMP] for the α1 and α2 AMPK isoforms yields an A0.5 equal to 5.7 ± 2.0 and 16 ± 3.5 μm AMP, respectively (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar). Here, we measured total AMPK activity and, therefore, cannot distinguish the degree to which the individual isoforms are activated.The measurements of AMPK activity (without AMP) report the state activation of the AMPK cascade. Hardie et al. (14Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (315) Google Scholar) developed a model of the AMPK cascade using an A0.5 of 4 μm AMP that predicts full activation below 10 μm AMP. Our study, in accord with those of Hardieet al. (14Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (315) Google Scholar) and Stein et al. (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar), found a hyperbolic relationship between AMPK activity and [AMP]. Marsinet al. report a linear correlation between AMPK activity in the AMP/ATP range of 0.03–1.31 in perfused rat hearts using total AMP and ATP measured by high pressure liquid chromatography (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). Since most AMP is bound, however, the total AMP overestimates the cytosolic [AMP], which is presumably the allosteric regulator of the AMPK cascade activity. Our measurements of AMP/ATP are 100–1000 times lower than those reported by Marsin (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar).[ATP] and AMPKATP antagonizes the allosteric activation of AMPK by AMP because ATP competes with AMP for binding at the allosteric site but does not promote formation of the active conformation (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar). Because of this, Hardie and Carling have suggested that the kinase reacts to the AMP/ATP ratio (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar). Using our methods to alter myocardial energy metabolism, the AMP/ATP ratio ranged from 0.132 × 10−3 in the GBA-Bu group to 4.768 × 10−3 in the GBA450 group, with an AMP/ATP ratioA0.5 of 0.210 × 10−3. The model of the AMPK cascade by Hardie et al. predicts that theA0.5 of 4 μm with 0.2 mm ATP will increase to 40 μm at 2.0 mm ATP (14Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (315) Google Scholar). In vitro measurements of AMPK activity report an A0.5 of 4.4 ± 2.2 μm AMP at 0.2 mm ATP, but with 4 mm ATP, the A0.5 was 29 ± 14 μm (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar). The A0.5 of 1.8 μm in the present study reports on the activation of the AMPK cascade in vivo. This implies a lowA0.5 for AMPK phosphorylation in vivo, where [ATP] was always greater than 7 mm.[H+] and AMPKIntracellular [H+] may alter AMPK activity. A study of AMPK in vitro found that progressive decreases of pH from 7.3 to 6.6 resulted in progressive increases in AMPK activity (31Ponticos M. Lu Q.L. Morgan J.E. Hardie D.G. Partridge T.A. Carling D. EMBO J. 1998; 17: 1688-1699Crossref PubMed Scopus (274) Google Scholar). Manipulating energy metabolism in this study only modestly altered pHi. The GBA group hearts have a pHi of 7.0 when paced at 300 bpm and 6.9 when paced at 450 bpm, both of which are lower than the 7.1 of the Glc group. Thus, the alterations of AMPK activity observed in this study occurred at near normal pHi. Myocardial pHi typically decreases well below 7 during ischemia. If increased [H+] alters AMPK activity, this may contribute to the greater AMPK activity in the ischemic heart.AMPK and ACCAMPK activity plays a major role in the regulation, by phosphorylation, of ACC activity in the heart (28Dyck J.R. Kudo N. Barr A.J. Davies S.P. Hardie D.G. Lopaschuk G.D. Eur. J. Biochem. 1999; 262: 184-190Crossref PubMed Scopus (135) Google Scholar) and skeletal muscle (12Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar). ACC catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which plays a pivotal role in the regulation of fatty acid metabolism. In heart and skeletal muscle, malonyl-CoA regulates carnitine palmitoyltransferase-1. Carnitine palmitoyltransferase-1 transfers long chain fatty acids from the cytosol into the mitochondrion, where the fatty acids are oxidized by β-oxidation. In the rat heart during reperfusion following ischemia, increased AMPK activity correlates with reduced ACC activity (32Kudo N. Gillespie J.G. Kung L. Witters L.A. Schulz R. Clanachan A.S. Lopaschuk G.D. Biochim. Biophys. Acta. 1996; 1301: 67-75Crossref PubMed Scopus (214) Google Scholar). In this study, we also found a negative correlation between ACC activity and AMPK activity.ConclusionsOur findings reveal important details of AMPK regulation in the heart. Because of its activation by AMP, AMPK is hypothesized to act as a "low fuel warning system" (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar) or as a "master switch" for cellular energy levels (33Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). For AMPK to function as a metabolic sensor, it must be activated rapidly and early during a metabolic stress if it is to help sustain energy levels. Our results indicate that the A0.5 for activation of AMPK was 1.8 μm. AMP concentrations in that range can result from PCr decreases of about 30% or less in the heart. These characteristics are consistent with AMPK functioning as a low fuel warning system. These results also indicate that AMPK activation could conceivably occur in situations other than ischemia. For example, Neubauer et al. (34Neubauer S. Horn M. Cramer M. Harre K. Newell J.B. Peters W. Pabst T. Ertl G. Hahn D. Ingwall J.S. Kochsiek K. Circulation. 1997; 96: 2190-2196Crossref PubMed Scopus (581) Google Scholar) reported 30% decreases in the PCr/ATP ratio in the human heart during failure.The measures of PCr, ATP, and pHi by 31P NMR provide the most accurate estimates of cytosolic [AMP] available. This enabled us to measure in the intact heart anA0.5 of 1.8 μm AMP, which is comparable with the A0.5 measured in vitro in the presence of 0.2 mm ATP. In vitro, the AMPK A0.5 increased 10–20-fold at 4 mm ATP due to the antagonizing effect of ATP on the AMP activation of AMPK. In the present study, [ATP] was, however, always greater than 7 mm. The lowA0.5 in the presence of 7 mm [ATP] in the intact heart suggests several possible features of AMPK regulation in vivo: first, the ATP antagonism of AMPK activation is reduced; second, AMPK senses an [ATP] that is different than the average cytosolic concentration; or third, factors in addition to AMP activate AMPK in the heart. These possibilities remain to be explored. AMP-activated protein kinase (AMPK)1 and AMPK kinase comprise a protein kinase cascade that has been highly conserved throughout evolution (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar, 2Hardie D.G. Carling D. Carlson M. Ann. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1266) Google Scholar). Increases in AMP concentration ([AMP]) activate this cascade by four mechanisms (3Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 4Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (492) Google Scholar, 5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar). These mechanisms are as follows: 1) an allosteric activation by AMP of AMPK kinase, which then phosphorylates AMPK; 2) the binding of AMP to AMPK, which makes it a poorer substrate for protein phosphatases; 3) the binding of AMP to AMPK, which makes AMPK a better substrate for AMPK kinase; and 4) the allosteric activation by AMP of AMPK. The activating effects of AMP are antagonized by high concentrations of ATP. Since the AMPK is activated when AMP is elevated and ATP is depressed, AMPK is hypothesized to act as cellular "fuel gauge" (1Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1126) Google Scholar). After AMPK activation, in response to metabolic stress, AMPK phosphorylates enzymes, leading to activation of catabolic pathways to increase ATP synthesis and inhibition of anabolic pathways to limit ATP consumption. For example, AMPK phosphorylation decreases acetyl-CoA-carboxylase (ACC) activity, which decreases malonyl-CoA concentration. Malonyl-CoA inhibits carnitine palmitoyltransferase-1, which transports fatty acids into the mitochondrion (6Lopaschuk G.D. Am. J. Cardiol. 1997; 80: 11A-16AAbstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 7Muoio D.M. Seefeld K. Witters L.A. Coleman R.A. Biochem. J. 1999; 338: 783-791Crossref PubMed Scopus (342) Google Scholar). Reduction of malonyl-CoA increases fatty acid uptake via carnitine palmitoyltransferase-1 and, thereby, the fatty acid oxidation by the mitochondria, which increases ATP production. In skeletal muscle and hearts, AMPK activity also increases glucose uptake by enhancing GLUT-4 translocation (8Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Crossref PubMed Scopus (583) Google Scholar, 9Hayashi T. Hirshman M.F. Fujii N. Habinowski S.A. Witters L.A. Goodyear L.J. Diabetes. 2000; 49: 527-531Crossref PubMed Scopus (379) Google Scholar, 10Russell III, R.R. Bergeron R. Shulman G.I. Young L.H. Am. J. Physiol. 1999; 277: H643-H649PubMed Google Scholar). In hearts, AMPK phosphorylation of 6-phosphofructo-2-kinase increases fructose 2,6-bisphosphate, a stimulator of 6-phosphofructo-1-kinase, which accelerates glycolysis (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). AMPK also down-regulates ATP-consuming pathways, such as glycogen, cholesterol, and fatty acid synthesis (12Winder W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar, 13Vavvas D. Apazidis A. Saha A.K. Gamble J. Patel A. Kemp B.E. Witters L.A. Ruderman N.B. J. Biol. Chem. 1997; 272: 13255-13261Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Several studies have examined the relationship between the activation of AMPK and [AMP]. Hardie et al. (14Hardie D.G. Salt I.P. Hawley S.A. Davies S.P. Biochem. J. 1999; 338: 717-722Crossref PubMed Scopus (315) Google Scholar) modeled the AMPK cascade. Their model predicts a sigmoidal response of AMPK activity to increasing [AMP] with a half-maximal activation (A0.5) of 4 μm. Stein et al. measured AMPK activity of the α1 and α2 isoforms in vitro as a function of [AMP] (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar). These measurements showed a hyperbolic increase in AMPK activity with increasing [AMP], with an A0.5 of 5.7 μm for α1 and 16 μm for α2. In contrast, Marsin et al. (11Marsin A.S. Bertrand L. Rider M.H. Deprez J. Beauloye C. Vincent M.F. Van den Berghe G. Carling D. Hue L. Curr. Biol. 2000; 10: 1247-1255Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar) reported a linear relationship between AMPK activity and AMP/ATP ratio, using total AMP measured in tissue extracts of isolated rat hearts during ischemia or treatment with inhibitors of ATP synthesis. The aim of the present study was to define the relationship between AMPK activation and cytosolic [AMP] in vivo in the isolated perfused rat heart. To accomplish this, we employed an approach that restrains substrate metabolism by use of the inhibitors bromo-octanoate (BrO) to inhibit fatty acid oxidation (16Schulz H. Life Sci. 1987; 40: 1443-1449Crossref PubMed Scopus (52) Google Scholar) and amino-oxyacetate (AOA), to inhibit pyruvate oxidation (17Safer B. Smith C.M. Williamson J. J. Mol. Cell Cardiol. 1971; 2: 111-124Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Using this approach together with increased work demand reduced the phosphocreatine (PCr) (18Balschi J.A. Shen H. Madden M.C. Hai J.O. Bradley Jr., E.L. Wolkowicz P.E. J. Mol. Cell. Cardiol. 1997; 29: 3123-3133Abstract Full Text PDF PubMed Scopus (13) Google Scholar). We used 31P NMR spectroscopy to measure the PCr and ATP content as well as the intracellular pH (pHi) of these hearts. The creatine kinase and the adenylate kinase equilibrium expressions were used to calculate [ADP] and [AMP], respectively. The net effect of [PCr] reductions is an increase in [AMP] with a relatively constant [ATP]. Cytosolic [AMP] was therefore manipulated in a relatively controlled manner, and the AMPK activity was measured. DISCUSSIONResults in this paper define the relationship between AMPK activity and the cytosolic [AMP] in the isolated perfused rat heart. To our knowledge, this is the first correlation of AMPK activity with metabolically active cytosolic [AMP] in the isolated heart. To increase the cytosolic [AMP] in isolated perfused rat hearts, the heart's supply of acetyl-CoA was limited using the inhibitors bromo-octanoate and amino-oxyacetate. This method decreases both aerobic energy production and high energy phosphates, particularly PCr (18Balschi J.A. Shen H. Madden M.C. Hai J.O. Bradley Jr., E.L. Wolkowicz P.E. J. Mol. Cell. Cardiol. 1997; 29: 3123-3133Abstract Full Text PDF PubMed Scopus (13) Google Scholar). Because of the equilibrium of the creatine kinase reaction, the reduction of [PCr] results in an increase in [ADP]. In turn, the near equilibrium of the adenylate kinase reaction translates the increase in [ADP] into an increase in [AMP]. 31P NMR-measured PCr, ATP, and pHi provided an estimate of cytosolic [AMP] in vivo.Four conclusions can be drawn from the AMPK activity and [AMP] measurements of the seven groups of hearts studied here. First, Glc hearts, oxidizing both exogenous and endogenous glucose and endogenous triglycerides, maintain low values of [AMP] and AMPK activity. Second, AMPK activity in GBA-Bu hearts treated with inhibitors of fatty acid and glucose oxidation but provided with the substrate butyrate, which can bypass the blockade of β-oxidation, is equal to that of the Glc group hearts. These hearts maintain [AMP] equal to those of Glc hearts. These results show that AMPK activity does not increase due to nonspecific effects of the inhibitors. Third, AMPK activity increased in the GA hearts paced at 450 bpm. Fourth, GBA hearts treated with BrO and AOA to inhibit fatty acid and glucose oxidation have increased [AMP] as well as increased AMPK activity. In this study, hearts with an [AMP] above 10 μm demonstrate maximal AMPK activity (Fig. 2).Maximal increases in AMPK activity have been reported by a number of investigators. In vitro measurements by Stein found a 300% maximal activity for the α1 AMPK isoform and a 1300% maximal activity for the α2 AMPK isoform (15Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar). The heart contains both AMPK isoforms with the α2 isoform accounting for 70–80% of the total (27Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Wit
Referência(s)