Fatty Acids Attenuate Insulin Regulation of 5'-AMP–Activated Protein Kinase and Insulin Cardioprotection After Ischemia
http://www.100md.com
Clifford D.L. Folmes, Alexander S. Clana
参见附件。
the Cardiovascular Research Group and Departments of Pediatrics (C.D.L.F., G.D.L.) and Pharmacology (C.D.L.F., A.S.C.), University of Alberta, Edmonton, Canada.
Abstract
The cardioprotective effect of insulin during ischemia–reperfusion has been associated with stimulation of glucose uptake and glycolysis. Although fatty acids and 5'-AMP activated protein kinase (AMPK) are regulators of glucose metabolism, it is unknown what effect insulin has on postischemic function and AMPK activity in the presence of high levels of fatty acid. Isolated ejecting mouse hearts were perfused with Krebs–Henseleit solution containing 5 mmol · L–1 glucose and 0, 0.2, or 1.2 mmol · L–1 palmitate, with or without 100 μU/mL insulin. During aerobic perfusion in the absence of palmitate, insulin stimulated glycolysis by 73% and glucose oxidation by 54%, while inhibiting AMPK activity by 43%. In the presence of 0.2 or 1.2 mmol · L–1 palmitate, insulin stimulated glycolysis by 111% and 105% and glucose oxidation by 72% and 274% but no longer inhibited AMPK activity. During reperfusion of hearts in the absence of palmitate, insulin increased recovery of cardiac power by 47%. This was associated with a 97% increase in glycolysis and a 160% increase in glucose oxidation. However, in the presence of 1.2 mmol · L–1 palmitate, insulin now decreased recovery of cardiac power by 42%. During reperfusion, glucose oxidation was inhibited by high fat, but insulin-stimulated glycolysis remained high, resulting in increased proton production. In the absence of fatty acids, insulin blunted the ischemia-induced activation of AMPK, but this effect was lost in the presence of fatty acids. We demonstrate that the cardioprotective effect of insulin and its ability to inhibit AMPK activity are lost in the presence of high concentrations of fatty acids.
Key Words: fatty acid oxidation glucose oxidation glycolysis proton production ischemia–reperfusion
Introduction
The cardioprotective effect of insulin as part of glucose–insulin–potassium solution (GIK) was originally identified by Sodi-Pallares et al to reduce the electrocardiographic abnormalities associated with acute myocardial infarction (AMI).1 Recently, there has been renewed interest in the use of GIK therapy as a metabolic treatment for AMI because both a metaanalysis of previous trials and the ECLA (Estudios Cardiologicos Latinoamerica) trial showed that GIK significantly reduced proportional in-hospital mortality.2,3 However the results of this analysis have been recently questioned because of the CREATE (Clinical Trial of REviparin and Metabolic Modulation in Acute Myocardial Infarction Treatment Evaluation)–ECLA randomized controlled trial examining the effect of GIK in patients with ST-elevated myocardial infarction.4 Patients were assigned to receive either usual care or usual care with a high-dose GIK infusion regimen. That study showed no efficacy of GIK therapy in any of the predefined end points, including mortality, cardiac arrest, and cardiogenic shock. Despite the fact that insulin can suppress circulating fatty acid concentrations, plasma fatty acids levels were never assessed in that study. Therefore, the normal insulin-mediated decrease in plasma fatty acids may not have occurred, which could possibly account for the negative results.
The cardioprotective effect of insulin has been well established in experimental studies in which hearts have been perfused in the absence of fatty acids.5–7 This beneficial effect may be attributable either to a direct positive inotropic effect of insulin or to a metabolic effect of insulin, such as stimulation of anaerobic glycolysis to contribute to basal ATP production.
In addition to insulin, AMP-activated protein kinase (AMPK) is also an important regulator of glucose metabolism in the heart.8,9 AMPK acts as a cellular fuel gauge, by shutting off energy requiring processes and stimulating energy yielding processes during times of metabolic stress. We and others have shown that AMPK is rapidly activated during myocardial ischemia and is associated with dramatic changes in the control of glucose and fatty acid metabolism.10–12 This ischemia-induced activation of AMPK can mimic the effect of insulin on myocardial glucose uptake and glycolysis.12,13 However, unlike insulin, AMPK activation is associated with an acceleration of fatty acid oxidation in the heart, which, in turn, leads to a decrease in glucose oxidation.14 Of potential importance is that we and others have demonstrated that insulin can inhibit the activation of AMPK during both aerobic perfusion and ischemia in the presence or absence of normal concentrations of fatty acids (0.4 mmol · L–1 palmitate bound to 3% BSA or 0 mmol · L–1 palmitate, respectively); however, recovery of postischemic function was not assessed in these studies. Therefore, insulin may have a salutary effect in ischemia by indirectly inhibiting fatty acid oxidation.
Under normal aerobic conditions, the heart preferentially metabolizes fatty acids, which contribute between 60% and 80% of the energy requirements of the heart. However, during and following most clinically relevant forms of myocardial ischemia, the heart is exposed to high levels of circulating fatty acids.15 When hearts are reperfused following severe ischemia, fatty acid oxidation quickly recovers and predominates as the main source of mitochondrial oxidative metabolism.16–18 This is attributable both to the exposure of the heart to a high concentration of fatty acids as well as to subcellular changes in the control of fatty acid oxidation.14 During ischemia, anaerobic glycolysis increases and becomes an important source of ATP production that has a role in the maintenance of ion gradients in the cardiomyocyte.19 However, if the pyruvate from glycolysis is not subsequently oxidized, such as in the presence of high fatty acids, there is a production of both lactate and protons, the latter produced by the hydrolysis of glycolytically derived ATP. This proton production is a major contributor to the intracellular acidosis that is associated with ischemia.20 The acidosis can lead to a sequelae of adverse events, including accelerated sarcolemmal Na+/H+ exchange leading to intracellular Na+ and Ca2+ overload,21,22 decreased cardiac pressure development,3 the initiation of cardiac arrhythmias,23 and decreased response of contractile proteins to Ca2+.23 If the pyruvate from glycolysis is aerobically metabolized (ie, glucose oxidation), then lactate and protons are not produced.24,25
Despite the importance of insulin, AMPK, and fatty acids in controlling glucose metabolism in the heart, little is known about their interaction during and following ischemia. Recently, Clark et al showed that fatty acids can attenuate the ability of insulin to inhibit AMPK activation.26 Because fatty acids are elevated in most clinical forms of myocardial ischemia, it is possible that high fatty acids overcome any beneficial effects of insulin during ischemia.27 Therefore, we investigated the effect of insulin on AMPK activation and myocardial metabolism in the presence of graded concentrations of fatty acids. In addition, we considered the implications of these observations on AMPK activation during ischemia and the effect on recovery of contractile function following ischemia.
Materials and Methods
Animals
The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with Canadian Council of Animal Care guidelines.
Isolated Ejecting Mouse Heart Model
CD-1 mouse hearts (98 mice) were perfused in the working ejecting mode as previously described.28 Briefly, mice were anesthetized with pentobarbital sodium (12 mg IP), and the hearts were subsequently excised and cannulated via the aorta and left atrium. After equilibration in the Langendorff mode, hearts were switched to the ejecting mode and perfused with modified Krebs–Henseleit solution containing 118.5 mmol · L–1 NaCl, 25 mmol · L–1 NaHCO3, 4.7 mmol · L–1 KCl, 1.2 mmol · L–1 MgSO4, 1.2 mmol · L–1 KH2PO4, 2.5 mmol · L–1 CaCl2, 0.5 mmol · L–1 EDTA, 5 mmol · L–1 [5-3H/U-14C]glucose, and 3% fatty acid–free albumin. Hearts were perfused in the presence or absence of 100 μU/mL insulin, depending on the experimental protocol. The oxygenated Krebs–Henseleit solution contained either no fatty acids (no fat) or 0.2 (low fat) or 1.2 mmol · L–1 (high fat) palmitate. The palmitate was prebound to the albumin as described previously.29 Glycolysis and glucose oxidation were measured by quantitative collection of the 3H2O and 14CO2, respectively, derived from [5-3H/U-14C]glucose, as described previously (see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org).24,29
Spontaneously beating hearts were perfused at a constant left atrial preload pressure of 11.5 mm Hg and a constant aortic afterload pressure of 50 mm Hg for: (1) a 40-minute aerobic perfusion; (2) a 30-minute aerobic perfusion followed by 16 minutes (no fat) or 18 minutes (high fat) of global no-flow ischemia and 40 minutes of aerobic reperfusion; (3) a 30-minute aerobic control; and (4) a 30-minute aerobic perfusion followed by 16 minutes (no fat) or 18 minutes (high fat) of global no-flow ischemia, for an ischemia control (supplemental Figure I). At the end of the perfusion protocols, the hearts were quick frozen in liquid nitrogen with Wollenberger tongs and stored at –80°C.
Tissue Preparation for AMPK Activity Assay
Approximately 20 mg of frozen ventricular tissue was homogenized for 30 seconds using a Polytron Homogenizer in 80 μL of homogenization buffer containing 50 mmol · L–1 Tris-HCl (pH 8 at 4°C), 1 mmol · L–1 EDTA, 10% (wt/vol) glycerol, 0.02% (vol/vol) Brij-35, 1 mmol · L–1 dithiothreitol (DTT), protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). The homogenates were centrifuged at 10 000g for 20 minutes at 4°C, and the resultant supernatant was used to assay AMPK activity, as previously described, except the synthetic AMARA peptide was used as the substrate.11
Statistical Analysis
All data are presented as the mean±SEM. The data were analyzed with the statistical program Instat 2.01 and GB-stat. Two-way repeated-measures ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for cardiac power. One-way or 2-way ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for the metabolic data. For the AMPK activity data, a t test was used to determine differences between the no insulin and insulin groups at the 3 different time points (aerobic, ischemia, and reperfusion) and then a 2-way ANOVA to determine the difference between the time points. Values of P<0.05 were considered significant.
Results
Baseline Aerobic Values in Forty-Minute Aerobic Hearts Perfused in the Absence of Fatty Acids
The addition of insulin to aerobically perfused mouse hearts in the absence of fatty acids caused a significant increase in cardiac power (71±4 to 87±6 mJ · min–1, P<0.05, for the no-insulin and insulin groups, respectively). Heart rate, cardiac output, and coronary flow did not differ between these groups (data not shown). As expected, insulin also increased glucose metabolism in these hearts, resulting in a 73% increase in glycolysis (Figure 1B; P<0.05) and a 54% increase in glucose oxidation (Figure 1C; P<0.05). Insulin also resulted in a 43% decrease in AMPK activity (Figure 1A; P<0.05).
Immunoblot analysis showed that insulin produces a 3-fold increase in phosphorylation of Akt at Ser473 (supplemental Figure IIA). Despite this activation of Akt, there was no downstream effect on phosphorylation of AMPK at Ser485/491 (supplemental Figure IIB).
Baseline Aerobic Values in Forty-Minute Aerobic Hearts Perfused in the Presence of Fatty Acids
The addition of insulin to perfused mouse hearts in the presence of either 0.2 or 1.2 mmol · L–1 palmitate did not significantly modify cardiac power or any of the other parameters of mechanical function (data not shown). Despite the lack of change in mechanical function, insulin resulted in a 111% increase in glycolysis in the low-fat group (Figure 2B; P<0.05) and a 106% increase in glycolysis in the high-fat group (Figure 2B; P<0.05). Insulin also increased glucose oxidation by 72% in the low-fat group (Figure 2B; P<0.05) and by 273% in the high-fat group (Figure 2C; P<0.05). However, in the presence of both low and high fat, insulin no longer inhibited AMPK activity (Figure 2A).
Immunoblot analysis also showed that in the presence of high fat, insulin produces a 2.5-fold increase in phosphorylation of Akt at Ser473 (supplemental Figure IA) and, as seen in the no fat hearts, there is no downstream effect on phosphorylation of AMPK at Ser485/491 (supplemental Figure IB).
Effect of Insulin and Palmitate on Myocardial Metabolism and Functional Recovery During Reperfusion Following Global No-Flow Ischemia
Ischemia and Reperfusion in the Absence of Fatty Acids
Insulin produced a small but insignificant increase in cardiac output and cardiac power and did not significantly change any other parameters of mechanical function of hearts perfused in the absence of fatty acids during the first 30 minutes of aerobic perfusion (Figure 3 and supplemental Table I). Similar to the previous aerobically perfused group, insulin increased glycolysis by 73% (Figure 4A) and glucose oxidation by 45% during the initial aerobic period (Figure 4B).
During reperfusion of hearts following ischemia, insulin produced a 47% increase in the recovery of cardiac power in hearts perfused in the absence of fatty acids (Figure 3A and supplemental Table I; P<0.05). Insulin effects on glucose metabolism persisted during reperfusion, where a 97% increase in glycolysis was observed (Figure 4A; P<0.05), as well as a 160% increase in glucose oxidation (Figure 4B P<0.05). Glucose oxidation during reperfusion recovered to the corresponding preischemic value in both the presence and absence of insulin. During reperfusion, a slight reduction in glycolysis in the insulin-treated hearts was observed, although this did not reach statistical significance (P=0.08). Although proton production was increased by 130% by insulin during aerobic perfusion (Figure 4C; P<0.05), insulin had no significant effect on proton production during reperfusion (Figure 4C).
Ischemia and Reperfusion in the Presence of a High Concentration of Fatty Acids
During the initial aerobic period, the presence of insulin did not significantly alter any parameters of mechanical function (supplemental Table II; P<0.05) but produced a 105% increase in glycolysis (Figure 5A; P<0.05) and a 274% increase in glucose oxidation (Figure 5B). However, during reperfusion in the presence of high fat, the cardioprotective effect of insulin seen in the absence of fatty acids was lost, and insulin now impaired recovery of cardiac power by 45%. (Figure 3B and supplemental Table II; P<0.05). During reperfusion, insulin increased glycolysis by 91% (Figure 5A; P<0.05) and glucose oxidation by 67% (Figure 5B; P<0.05). Unlike the no-fat group, glycolysis remained elevated during reperfusion, and glucose oxidation was suppressed by 44% compared with aerobic values (Figure 5B; P<0.05). As a consequence, proton production from glucose metabolism was increased by 92% in the insulin-treated hearts during reperfusion, compared with the no-insulin hearts (Figure 5C; P<0.05). The addition of insulin suppressed rates of fatty acid oxidation during both the aerobic period and the reperfusion period (Figure 5D; P<0.05).
Ischemia and Reperfusion Effects on AMPK Activity
In hearts perfused in the absence of fatty acids and frozen immediately following ischemia, there was a dramatic increase in AMPK activity (Figure 6A; P<0.05) that was inhibited by insulin (Figure 6A; P<0.05).
In the hearts perfused with high fat, insulin did not have any effect on AMPK activity either during the aerobic perfusion or postischemic reperfusion (Figure 6B). In addition, insulin was no longer able to blunt the dramatic rise in AMPK activity that occurred during ischemia in the presence of high fat (Figure 6B).
Discussion
A number of previous studies have examined the importance of substrate preference and its relationship to the recovery of cardiac function during reperfusion of severely ischemic hearts.16–18,24,30,31 Although insulin, fatty acids, and AMPK all have important effects on both function and metabolism following ischemia, the interaction among these 3 regulators of metabolism has not been examined. In this study, we used the isolated ejecting mouse heart, which is very sensitive to insulin, to examine the effect of insulin and fatty acids on myocardial metabolism, AMPK activity, and functional recovery during reperfusion following ischemia. Using this approach, we made a number of important findings. First, the ability of insulin to inhibit AMPK activity during both aerobic perfusion and during ischemia is dependent on the presence of fatty acids in the perfusate. Second, the cardioprotective effect of insulin is lost when hearts are perfused in the presence of high fatty acid levels that are normally seen during and following clinically relevant ischemia. Third, although insulin cannot inhibit AMPK activity in the presence of a high concentration of fatty acid, other downstream effectors of insulin are intact, including its ability to stimulate glycolysis and glucose oxidation.
A recent study by Clark et al examined the effect of fatty acids on insulin inhibition of AMPK in aerobically perfused rat hearts.26 Those authors confirmed our previous observation that insulin can inhibit AMPK activity.32,33 However, they also observed that this effect was lost as fatty acid concentrations were increased. In this study, we reproduced the results in the perfused mouse heart, showing that insulin inhibits AMPK in the absence of fatty acids but loses this ability in the presence of low (0.2 mmol · L–1) or high (1.2 mmol · L–1) concentrations of palmitate (Figure 1 and 2). This high-fat concentration was chosen to reproduce clinically relevant conditions in humans, where ischemia results in an increased efflux of fatty acids into the serum causing a rapid elevation of circulating fatty acids. We have previously characterized this response in adult patients who had undergone an AMI, and free fatty acid concentrations peaked at 1.51±0.15 mmol · L–1 (compared with 0.40±0.013 mmol · L–1 in control subjects).27
A novel finding of our study is that even though palmitate attenuates the ability of insulin to inhibit AMPK, other downstream signaling pathways of insulin are intact, because insulin activation of glycolysis, glucose oxidation, and phosphorylation of Ser473 of Akt (Figures 1 and 2 and supplemental Figure IIA) was not affected by the presence of fatty acid. This suggests that, in mouse heart, there is a fatty acid–dependent insulin-signaling pathway that modifies AMPK activity and a fatty acid independent insulin-signaling pathway that modifies myocardial metabolism.
These observations are of particular importance, as it has been shown that insulin can inhibit the ischemia-induced activation of AMPK,34 which would benefit the ischemic and reperfused myocardium by decreasing fatty acid oxidation. However, these previous studies were performed in the absence of fatty acids. It has also been shown that during reperfusion of ischemic hearts oxidative metabolism quickly recovers, but high rates of fatty acid oxidation result in low glucose oxidation rates attributable to the Randle cycle.24,30,31,35 This reduction in glucose oxidation is detrimental during reperfusion as it contributes to the severity of acidosis in the ischemic and reperfused heart. Ischemia-induced activation of AMPK contributes to the low glucose oxidation rates during reperfusion of ischemic hearts, secondary to stimulation of fatty acid oxidation.10 Insulin may attenuate these effects by inhibiting AMPK. However, we observed that insulin only inhibited AMPK activity in hearts perfused in the absence of fatty acids. Although insulin only induces a 15% inhibition in AMPK activity at the end of ischemia, we may be missing a larger effect as Beauloye et al showed that ischemia-induced AMPK activation peaks after 10 minutes of global ischemia and decreases with extended ischemic times.34 Therefore, insulin may suppress AMPK activation to a greater extent at 10 minutes of ischemia where peak activation occurs.
The cardioprotective effect of insulin has been well established in experimental studies.5–7 It has been suggested that this effect is attributable to either a positive inotropic effect or to a metabolic effect of insulin, such as the stimulation of anaerobic glycolysis to maintain basal ATP production and to switch the preferred oxidative substrate to glucose, which is a more oxygen-efficient substrate and does not produce the toxic intermediates of fatty acid oxidation. As seen in Figure 3, we have confirmed that insulin produces a cardioprotective effect in the absence of fatty acids, improving recovery of cardiac power by 47%. The beneficial effect of insulin during ischemia and reperfusion in the absence of fatty acids may be attributable to an acceleration of glycolysis, which would increase the amount of ATP being produced by the heart. In addition, proton production from glucose metabolism was not significantly elevated by insulin in hearts perfused in the absence of fatty acids. Therefore, the beneficial effects of insulin would be 2-fold, by increasing ATP production by the stimulation of glycolysis and glucose oxidation.
A surprising finding from this study is that in the presence of high fat, insulin impaired the recovery of mechanical function during reperfusion. Like the no-fat condition, insulin stimulated both glycolysis and glucose oxidation during the aerobic period and the reperfusion period. However, because insulin produced a greater stimulation of glycolysis than glucose oxidation, there was an increased in proton production from glucose metabolism during reperfusion (Figure 5). This may explain the detrimental effects of insulin during high-fat perfusion.30,31 As rates of fatty acid oxidation increase, this increases intracellular concentrations of acetyl–coenzyme A (acetyl-CoA), which is an allosteric inhibitor of pyruvate dehydrogenase (PDH). Indeed, the failure of insulin to inhibit AMPK activity may also contribute to this phenomenon. AMPK activation may lead to an acceleration of glucose uptake, thus supplying substrate for glycolysis, but also leads to phosphorylation of acetyl-CoA carboxylase, leading to a decrease in malonyl-CoA levels and an acceleration of fatty acid oxidation, which suppresses glucose oxidation by the abovementioned mechanism (supplemental Table II).
However, proton production during reperfusion in the high-fat plus insulin group was not increased significantly from values in the no-fat plus insulin group, despite dramatic differences in the effect on insulin on postischemic contractile function. The benefits of insulin on recovery of contractile function in the no-fat hearts may be related to an increase in energy supply to these hearts. The no-fat group may be energy starved and energetically compromised; thus, energy supply would be a better determinant of postischemic contractile function than proton production. Insulin decreased the AMP/ATP ratio during aerobic perfusion and increased ATP at the end of reperfusion in the no-fat group (supplemental Table III). However, in the presence of high fat, insulin did not change the AMP/ATP ratio during either aerobic perfusion or during reperfusion.
During the preparation of this report, a study by Horman et al was published suggesting that insulin may inhibit AMPK via Akt phosphorylation of Ser485/491 on the subunit of AMPK, corresponding with a previous report that Akt activity negatively correlates with AMPK Thr172 phosphorylation.36,37 Contradictory results were obtained in the present study. The addition of insulin produced an increase in phosphorylation of Akt at Ser473, which is indicative of Akt activity. Despite this apparent activation of this insulin-signaling pathway, there was no downstream inhibition of AMPK in the no fat palmitate group. This difference may be attributable to the use of a supraphysiological insulin concentration of 100 nmol · L–1 in the Horman study, compared with a more physiological concentration (0.6 nmol · L–1) in the present study. The only clinical study to report arterial insulin concentrations after GIK administration was a study of coronary surgery patients with type 2 diabetes. High-dose GIK therapy increased insulin concentrations to 10.3 nmol · L–1 following administration.38
Considerable interest has also focused on GIK in both AMI and cardiac surgery patients.3,4 However, the recent large CREATE trial did not show GIK effectiveness. It is possible that the discrepant results may be related to differences in plasma fatty acid levels in the patient population. Despite the fact that some of the benefits of GIK have been attributed to a lowering of plasma free fatty acids, previous trials have not determined what effect GIK has on plasma fatty acids levels post–myocardial infarction or postsurgery. In addition, the time of administration of GIK may play a key role in its efficacy, as the only group in the CREATE-ECLA trial with a trend to improvement was the population that received GIK before percutaneous coronary intervention, and this did reach statistical significance in a more recent trial.4,39
In summary, this study demonstrates that the cardioprotective effect of insulin and its ability to modify AMPK activity in the isolated ejecting mouse heart is highly dependent on the concentration of fatty acids present in the perfusate. However, other insulin-signaling pathways, such as modification of glucose metabolism, are still intact in the presence of clinically relevant high concentrations of fatty acids. These findings have important implications in the use of insulin therapy for the treatment of ischemic heart disease.
Acknowledgments
Sources of Funding
Funded by a grant from the Canadian Institutes of Health Research. C.D.L.F. holds an Alberta Heritage for Medical Research (AHFMR) Studentship and a Natural Sciences and Engineering Research Council Scholarship. G.D.L. is an AHFMR Medical Scientist.
Disclosures
None.
Footnotes
Original received December 17, 2005; revision received May 10, 2006; accepted May 19, 2006.
References
Sodi-Pallares D, Ponce de LJ, Bisteni A, Medrano GA. Potassium, glucose, and insulin in myocardial infarction. Lancet. 1969; 1: 1315–1316. [Order article via Infotrieve]
Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction: an overview of randomized placebo-controlled trials. Circulation. 1997; 96: 1152–1156.
Diaz R, Paolasso EA, Piegas LS, Tajer CD, Moreno MG, Corvalan R, Isea JE, Romero G. Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) Collaborative Group. Circulation. 1998; 98: 2227–2234.
Mehta SR, Yusuf S, Diaz R, Zhu J, Pais P, Xavier D, Paolasso E, Ahmed R, Xie C, Kazmi K, Tai J, Orlandini A, Pogue J, Liu L. Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial. JAMA. 2005; 293: 437–446.
Doenst T, Richwine RT, Bray MS, Goodwin GW, Frazier OH, Taegtmeyer H. Insulin improves functional and metabolic recovery of reperfused working rat heart. Ann Thorac Surg. 1999; 67: 1682–1688.
Zaha V, Francischetti I, Doenst T. Insulin improves postischemic recovery of function through PI3K in isolated working rat heart. Mol Cell Biochem. 2003; 247: 229–232. [Order article via Infotrieve]
Fischer-Rasokat U, Beyersdorf F, Doenst T. Insulin addition after ischemia improves recovery of function equal to ischemic preconditioning in rat heart. Basic Res Cardiol. 2003; 98: 329–336. [Order article via Infotrieve]
Winder WW, Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol. 1999; 277: E1–E10.
Hardie DG, Hawley SA. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays. 2001; 23: 1112–1119. [Order article via Infotrieve]
Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. 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. 1995; 270: 17513–17520.
Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, Lopaschuk GD. 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. 1996; 1301: 67–75. [Order article via Infotrieve]
Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den BG, Carling D, Hue L. Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr Biol. 2000; 10: 1247–1255. [Order article via Infotrieve]
Russell RR III, Bergeron R, Shulman GI, Young LH. Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol. 1999; 277: H643–H649.
Kantor PF, Dyck JR, Lopaschuk GD. Fatty acid oxidation in the reperfused ischemic heart. Am J Med Sci. 1999; 318: 3–14. [Order article via Infotrieve]
Lopaschuk GD, Belke DD, Gamble J, Itoi T, Schonekess BO. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta. 1994; 1213: 263–276. [Order article via Infotrieve]
Liedtke AJ, Demaison L, Eggleston AM, Cohen LM, Nellis SH. Changes in substrate metabolism and effects of excess fatty acids in reperfused myocardium. Circ Res. 1988; 62: 535–542.
Lerch R, Tamm C, Papageorgiou I, Benzi RH. Myocardial fatty acid oxidation during ischemia and reperfusion. Mol Cell Biochem. 1992; 116: 103–109. [Order article via Infotrieve]
Lopaschuk GD, Spafford MA, Davies NJ, Wall SR. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res. 1990; 66: 546–553.
Neely JR, Morgan HE. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annu Rev Physiol. 1974; 36: 413–459.
Dennis SC, Gevers W, Opie LH. Protons in ischemia: where do they come from; where do they go to J Mol Cell Cardiol. 1991; 23: 1077–1086. [Order article via Infotrieve]
Karmazyn M, Moffat MP. Role of Na+/H+ exchange in cardiac physiology and pathophysiology: mediation of myocardial reperfusion injury by the pH paradox. Cardiovasc Res. 1993; 27: 915–924. [Order article via Infotrieve]
Scholz W, Albus U. Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion. Basic Res Cardiol. 1993; 88: 443–455. [Order article via Infotrieve]
Orchard CH, Houser SR, Kort AA, Bahinski A, Capogrossi MC, Lakatta EG. Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium. J Gen Physiol. 1987; 90: 145–165.
Lopaschuk GD, Wambolt RB, Barr RL. An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. J Pharmacol Exp Ther. 1993; 264: 135–144.
Opie LH. Myocardial ischemia-metabolic pathways and implications of increased glycolysis. Cardiovasc Drugs Ther. 1990; 4 (suppl 4): 777–790. [Order article via Infotrieve]
Clark H, Carling D, Saggerson D. Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids. Eur J Biochem. 2004; 271: 2215–2224.
Lopaschuk GD, Collins-Nakai R, Olley PM, Montague TJ, McNeil G, Gayle M, Penkoske P, Finegan BA. Plasma fatty acid levels in infants and adults after myocardial ischemia. Am Heart J. 1994; 128: 61–67. [Order article via Infotrieve]
Larsen TS, Belke DD, Sas R, Giles WR, Severson DL, Lopaschuk GD, Tyberg JV. The isolated working mouse heart: methodological considerations. Pflugers Arch. 1999; 437: 979–985. [Order article via Infotrieve]
Barr RL, Lopaschuk GD. Direct measurement of energy metabolism in the isolated working rat heart. J Pharmacol Toxicol Methods. 1997; 38: 11–17. [Order article via Infotrieve]
Liu B, Clanachan AS, Schulz R, Lopaschuk GD. Cardiac efficiency is improved after ischemia by altering both the source and fate of protons. Circ Res. 1996; 79: 940–948.
Liu Q, Docherty JC, Rendell JC, Clanachan AS, Lopaschuk GD. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J Am Coll Cardiol. 2002; 39: 718–725.
Gamble J, Lopaschuk GD. Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism. 1997; 46: 1270–1274. [Order article via Infotrieve]
Makinde AO, Gamble J, Lopaschuk GD. Upregulation of 5'-AMP-activated protein kinase is responsible for the increase in myocardial fatty acid oxidation rates following birth in the newborn rabbit. Circ Res. 1997; 80: 482–489.
Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, Hue L. Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett. 2001; 505: 348–352. [Order article via Infotrieve]
Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963; 1: 785–789. [Order article via Infotrieve]
Horman S, Vertommen D, Heath R, Neumann D, Mouton V, Woods A, Schlattner U, Wallimann T, Carling D, Hue L, Rider MH. Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase alpha-subunits in heart via hierarchical phosphorylation of Ser485/491. J Biol Chem. 2006; 281: 5335–5340.
Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JRB. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem. 2003; 278: 39422–39427.
Szabo Z, Arnqvist H, Hakanson E, Jorfeldt L, Svedjeholm R. Effects of high-dose glucose-insulin-potassium on myocardial metabolism after coronary surgery in patients with Type II diabetes. Clin Sci (Lond). 2001; 101: 37–43. [Order article via Infotrieve]
Yazici M, Demircan S, Durna K, Yasar E, Acar Z, Sahin M. Effect of glucose-insulin-potassium infusion on myocardial damage due to percutaneous coronary revascularization. Am J Cardiol. 2005; 96: 1517–1520. [Order article via Infotrieve]
the Cardiovascular Research Group and Departments of Pediatrics (C.D.L.F., G.D.L.) and Pharmacology (C.D.L.F., A.S.C.), University of Alberta, Edmonton, Canada.
Abstract
The cardioprotective effect of insulin during ischemia–reperfusion has been associated with stimulation of glucose uptake and glycolysis. Although fatty acids and 5'-AMP activated protein kinase (AMPK) are regulators of glucose metabolism, it is unknown what effect insulin has on postischemic function and AMPK activity in the presence of high levels of fatty acid. Isolated ejecting mouse hearts were perfused with Krebs–Henseleit solution containing 5 mmol · L–1 glucose and 0, 0.2, or 1.2 mmol · L–1 palmitate, with or without 100 μU/mL insulin. During aerobic perfusion in the absence of palmitate, insulin stimulated glycolysis by 73% and glucose oxidation by 54%, while inhibiting AMPK activity by 43%. In the presence of 0.2 or 1.2 mmol · L–1 palmitate, insulin stimulated glycolysis by 111% and 105% and glucose oxidation by 72% and 274% but no longer inhibited AMPK activity. During reperfusion of hearts in the absence of palmitate, insulin increased recovery of cardiac power by 47%. This was associated with a 97% increase in glycolysis and a 160% increase in glucose oxidation. However, in the presence of 1.2 mmol · L–1 palmitate, insulin now decreased recovery of cardiac power by 42%. During reperfusion, glucose oxidation was inhibited by high fat, but insulin-stimulated glycolysis remained high, resulting in increased proton production. In the absence of fatty acids, insulin blunted the ischemia-induced activation of AMPK, but this effect was lost in the presence of fatty acids. We demonstrate that the cardioprotective effect of insulin and its ability to inhibit AMPK activity are lost in the presence of high concentrations of fatty acids.
Key Words: fatty acid oxidation glucose oxidation glycolysis proton production ischemia–reperfusion
Introduction
The cardioprotective effect of insulin as part of glucose–insulin–potassium solution (GIK) was originally identified by Sodi-Pallares et al to reduce the electrocardiographic abnormalities associated with acute myocardial infarction (AMI).1 Recently, there has been renewed interest in the use of GIK therapy as a metabolic treatment for AMI because both a metaanalysis of previous trials and the ECLA (Estudios Cardiologicos Latinoamerica) trial showed that GIK significantly reduced proportional in-hospital mortality.2,3 However the results of this analysis have been recently questioned because of the CREATE (Clinical Trial of REviparin and Metabolic Modulation in Acute Myocardial Infarction Treatment Evaluation)–ECLA randomized controlled trial examining the effect of GIK in patients with ST-elevated myocardial infarction.4 Patients were assigned to receive either usual care or usual care with a high-dose GIK infusion regimen. That study showed no efficacy of GIK therapy in any of the predefined end points, including mortality, cardiac arrest, and cardiogenic shock. Despite the fact that insulin can suppress circulating fatty acid concentrations, plasma fatty acids levels were never assessed in that study. Therefore, the normal insulin-mediated decrease in plasma fatty acids may not have occurred, which could possibly account for the negative results.
The cardioprotective effect of insulin has been well established in experimental studies in which hearts have been perfused in the absence of fatty acids.5–7 This beneficial effect may be attributable either to a direct positive inotropic effect of insulin or to a metabolic effect of insulin, such as stimulation of anaerobic glycolysis to contribute to basal ATP production.
In addition to insulin, AMP-activated protein kinase (AMPK) is also an important regulator of glucose metabolism in the heart.8,9 AMPK acts as a cellular fuel gauge, by shutting off energy requiring processes and stimulating energy yielding processes during times of metabolic stress. We and others have shown that AMPK is rapidly activated during myocardial ischemia and is associated with dramatic changes in the control of glucose and fatty acid metabolism.10–12 This ischemia-induced activation of AMPK can mimic the effect of insulin on myocardial glucose uptake and glycolysis.12,13 However, unlike insulin, AMPK activation is associated with an acceleration of fatty acid oxidation in the heart, which, in turn, leads to a decrease in glucose oxidation.14 Of potential importance is that we and others have demonstrated that insulin can inhibit the activation of AMPK during both aerobic perfusion and ischemia in the presence or absence of normal concentrations of fatty acids (0.4 mmol · L–1 palmitate bound to 3% BSA or 0 mmol · L–1 palmitate, respectively); however, recovery of postischemic function was not assessed in these studies. Therefore, insulin may have a salutary effect in ischemia by indirectly inhibiting fatty acid oxidation.
Under normal aerobic conditions, the heart preferentially metabolizes fatty acids, which contribute between 60% and 80% of the energy requirements of the heart. However, during and following most clinically relevant forms of myocardial ischemia, the heart is exposed to high levels of circulating fatty acids.15 When hearts are reperfused following severe ischemia, fatty acid oxidation quickly recovers and predominates as the main source of mitochondrial oxidative metabolism.16–18 This is attributable both to the exposure of the heart to a high concentration of fatty acids as well as to subcellular changes in the control of fatty acid oxidation.14 During ischemia, anaerobic glycolysis increases and becomes an important source of ATP production that has a role in the maintenance of ion gradients in the cardiomyocyte.19 However, if the pyruvate from glycolysis is not subsequently oxidized, such as in the presence of high fatty acids, there is a production of both lactate and protons, the latter produced by the hydrolysis of glycolytically derived ATP. This proton production is a major contributor to the intracellular acidosis that is associated with ischemia.20 The acidosis can lead to a sequelae of adverse events, including accelerated sarcolemmal Na+/H+ exchange leading to intracellular Na+ and Ca2+ overload,21,22 decreased cardiac pressure development,3 the initiation of cardiac arrhythmias,23 and decreased response of contractile proteins to Ca2+.23 If the pyruvate from glycolysis is aerobically metabolized (ie, glucose oxidation), then lactate and protons are not produced.24,25
Despite the importance of insulin, AMPK, and fatty acids in controlling glucose metabolism in the heart, little is known about their interaction during and following ischemia. Recently, Clark et al showed that fatty acids can attenuate the ability of insulin to inhibit AMPK activation.26 Because fatty acids are elevated in most clinical forms of myocardial ischemia, it is possible that high fatty acids overcome any beneficial effects of insulin during ischemia.27 Therefore, we investigated the effect of insulin on AMPK activation and myocardial metabolism in the presence of graded concentrations of fatty acids. In addition, we considered the implications of these observations on AMPK activation during ischemia and the effect on recovery of contractile function following ischemia.
Materials and Methods
Animals
The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with Canadian Council of Animal Care guidelines.
Isolated Ejecting Mouse Heart Model
CD-1 mouse hearts (98 mice) were perfused in the working ejecting mode as previously described.28 Briefly, mice were anesthetized with pentobarbital sodium (12 mg IP), and the hearts were subsequently excised and cannulated via the aorta and left atrium. After equilibration in the Langendorff mode, hearts were switched to the ejecting mode and perfused with modified Krebs–Henseleit solution containing 118.5 mmol · L–1 NaCl, 25 mmol · L–1 NaHCO3, 4.7 mmol · L–1 KCl, 1.2 mmol · L–1 MgSO4, 1.2 mmol · L–1 KH2PO4, 2.5 mmol · L–1 CaCl2, 0.5 mmol · L–1 EDTA, 5 mmol · L–1 [5-3H/U-14C]glucose, and 3% fatty acid–free albumin. Hearts were perfused in the presence or absence of 100 μU/mL insulin, depending on the experimental protocol. The oxygenated Krebs–Henseleit solution contained either no fatty acids (no fat) or 0.2 (low fat) or 1.2 mmol · L–1 (high fat) palmitate. The palmitate was prebound to the albumin as described previously.29 Glycolysis and glucose oxidation were measured by quantitative collection of the 3H2O and 14CO2, respectively, derived from [5-3H/U-14C]glucose, as described previously (see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org).24,29
Spontaneously beating hearts were perfused at a constant left atrial preload pressure of 11.5 mm Hg and a constant aortic afterload pressure of 50 mm Hg for: (1) a 40-minute aerobic perfusion; (2) a 30-minute aerobic perfusion followed by 16 minutes (no fat) or 18 minutes (high fat) of global no-flow ischemia and 40 minutes of aerobic reperfusion; (3) a 30-minute aerobic control; and (4) a 30-minute aerobic perfusion followed by 16 minutes (no fat) or 18 minutes (high fat) of global no-flow ischemia, for an ischemia control (supplemental Figure I). At the end of the perfusion protocols, the hearts were quick frozen in liquid nitrogen with Wollenberger tongs and stored at –80°C.
Tissue Preparation for AMPK Activity Assay
Approximately 20 mg of frozen ventricular tissue was homogenized for 30 seconds using a Polytron Homogenizer in 80 μL of homogenization buffer containing 50 mmol · L–1 Tris-HCl (pH 8 at 4°C), 1 mmol · L–1 EDTA, 10% (wt/vol) glycerol, 0.02% (vol/vol) Brij-35, 1 mmol · L–1 dithiothreitol (DTT), protease inhibitors (Sigma), and phosphatase inhibitors (Sigma). The homogenates were centrifuged at 10 000g for 20 minutes at 4°C, and the resultant supernatant was used to assay AMPK activity, as previously described, except the synthetic AMARA peptide was used as the substrate.11
Statistical Analysis
All data are presented as the mean±SEM. The data were analyzed with the statistical program Instat 2.01 and GB-stat. Two-way repeated-measures ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for cardiac power. One-way or 2-way ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for the metabolic data. For the AMPK activity data, a t test was used to determine differences between the no insulin and insulin groups at the 3 different time points (aerobic, ischemia, and reperfusion) and then a 2-way ANOVA to determine the difference between the time points. Values of P<0.05 were considered significant.
Results
Baseline Aerobic Values in Forty-Minute Aerobic Hearts Perfused in the Absence of Fatty Acids
The addition of insulin to aerobically perfused mouse hearts in the absence of fatty acids caused a significant increase in cardiac power (71±4 to 87±6 mJ · min–1, P<0.05, for the no-insulin and insulin groups, respectively). Heart rate, cardiac output, and coronary flow did not differ between these groups (data not shown). As expected, insulin also increased glucose metabolism in these hearts, resulting in a 73% increase in glycolysis (Figure 1B; P<0.05) and a 54% increase in glucose oxidation (Figure 1C; P<0.05). Insulin also resulted in a 43% decrease in AMPK activity (Figure 1A; P<0.05).
Immunoblot analysis showed that insulin produces a 3-fold increase in phosphorylation of Akt at Ser473 (supplemental Figure IIA). Despite this activation of Akt, there was no downstream effect on phosphorylation of AMPK at Ser485/491 (supplemental Figure IIB).
Baseline Aerobic Values in Forty-Minute Aerobic Hearts Perfused in the Presence of Fatty Acids
The addition of insulin to perfused mouse hearts in the presence of either 0.2 or 1.2 mmol · L–1 palmitate did not significantly modify cardiac power or any of the other parameters of mechanical function (data not shown). Despite the lack of change in mechanical function, insulin resulted in a 111% increase in glycolysis in the low-fat group (Figure 2B; P<0.05) and a 106% increase in glycolysis in the high-fat group (Figure 2B; P<0.05). Insulin also increased glucose oxidation by 72% in the low-fat group (Figure 2B; P<0.05) and by 273% in the high-fat group (Figure 2C; P<0.05). However, in the presence of both low and high fat, insulin no longer inhibited AMPK activity (Figure 2A).
Immunoblot analysis also showed that in the presence of high fat, insulin produces a 2.5-fold increase in phosphorylation of Akt at Ser473 (supplemental Figure IA) and, as seen in the no fat hearts, there is no downstream effect on phosphorylation of AMPK at Ser485/491 (supplemental Figure IB).
Effect of Insulin and Palmitate on Myocardial Metabolism and Functional Recovery During Reperfusion Following Global No-Flow Ischemia
Ischemia and Reperfusion in the Absence of Fatty Acids
Insulin produced a small but insignificant increase in cardiac output and cardiac power and did not significantly change any other parameters of mechanical function of hearts perfused in the absence of fatty acids during the first 30 minutes of aerobic perfusion (Figure 3 and supplemental Table I). Similar to the previous aerobically perfused group, insulin increased glycolysis by 73% (Figure 4A) and glucose oxidation by 45% during the initial aerobic period (Figure 4B).
During reperfusion of hearts following ischemia, insulin produced a 47% increase in the recovery of cardiac power in hearts perfused in the absence of fatty acids (Figure 3A and supplemental Table I; P<0.05). Insulin effects on glucose metabolism persisted during reperfusion, where a 97% increase in glycolysis was observed (Figure 4A; P<0.05), as well as a 160% increase in glucose oxidation (Figure 4B P<0.05). Glucose oxidation during reperfusion recovered to the corresponding preischemic value in both the presence and absence of insulin. During reperfusion, a slight reduction in glycolysis in the insulin-treated hearts was observed, although this did not reach statistical significance (P=0.08). Although proton production was increased by 130% by insulin during aerobic perfusion (Figure 4C; P<0.05), insulin had no significant effect on proton production during reperfusion (Figure 4C).
Ischemia and Reperfusion in the Presence of a High Concentration of Fatty Acids
During the initial aerobic period, the presence of insulin did not significantly alter any parameters of mechanical function (supplemental Table II; P<0.05) but produced a 105% increase in glycolysis (Figure 5A; P<0.05) and a 274% increase in glucose oxidation (Figure 5B). However, during reperfusion in the presence of high fat, the cardioprotective effect of insulin seen in the absence of fatty acids was lost, and insulin now impaired recovery of cardiac power by 45%. (Figure 3B and supplemental Table II; P<0.05). During reperfusion, insulin increased glycolysis by 91% (Figure 5A; P<0.05) and glucose oxidation by 67% (Figure 5B; P<0.05). Unlike the no-fat group, glycolysis remained elevated during reperfusion, and glucose oxidation was suppressed by 44% compared with aerobic values (Figure 5B; P<0.05). As a consequence, proton production from glucose metabolism was increased by 92% in the insulin-treated hearts during reperfusion, compared with the no-insulin hearts (Figure 5C; P<0.05). The addition of insulin suppressed rates of fatty acid oxidation during both the aerobic period and the reperfusion period (Figure 5D; P<0.05).
Ischemia and Reperfusion Effects on AMPK Activity
In hearts perfused in the absence of fatty acids and frozen immediately following ischemia, there was a dramatic increase in AMPK activity (Figure 6A; P<0.05) that was inhibited by insulin (Figure 6A; P<0.05).
In the hearts perfused with high fat, insulin did not have any effect on AMPK activity either during the aerobic perfusion or postischemic reperfusion (Figure 6B). In addition, insulin was no longer able to blunt the dramatic rise in AMPK activity that occurred during ischemia in the presence of high fat (Figure 6B).
Discussion
A number of previous studies have examined the importance of substrate preference and its relationship to the recovery of cardiac function during reperfusion of severely ischemic hearts.16–18,24,30,31 Although insulin, fatty acids, and AMPK all have important effects on both function and metabolism following ischemia, the interaction among these 3 regulators of metabolism has not been examined. In this study, we used the isolated ejecting mouse heart, which is very sensitive to insulin, to examine the effect of insulin and fatty acids on myocardial metabolism, AMPK activity, and functional recovery during reperfusion following ischemia. Using this approach, we made a number of important findings. First, the ability of insulin to inhibit AMPK activity during both aerobic perfusion and during ischemia is dependent on the presence of fatty acids in the perfusate. Second, the cardioprotective effect of insulin is lost when hearts are perfused in the presence of high fatty acid levels that are normally seen during and following clinically relevant ischemia. Third, although insulin cannot inhibit AMPK activity in the presence of a high concentration of fatty acid, other downstream effectors of insulin are intact, including its ability to stimulate glycolysis and glucose oxidation.
A recent study by Clark et al examined the effect of fatty acids on insulin inhibition of AMPK in aerobically perfused rat hearts.26 Those authors confirmed our previous observation that insulin can inhibit AMPK activity.32,33 However, they also observed that this effect was lost as fatty acid concentrations were increased. In this study, we reproduced the results in the perfused mouse heart, showing that insulin inhibits AMPK in the absence of fatty acids but loses this ability in the presence of low (0.2 mmol · L–1) or high (1.2 mmol · L–1) concentrations of palmitate (Figure 1 and 2). This high-fat concentration was chosen to reproduce clinically relevant conditions in humans, where ischemia results in an increased efflux of fatty acids into the serum causing a rapid elevation of circulating fatty acids. We have previously characterized this response in adult patients who had undergone an AMI, and free fatty acid concentrations peaked at 1.51±0.15 mmol · L–1 (compared with 0.40±0.013 mmol · L–1 in control subjects).27
A novel finding of our study is that even though palmitate attenuates the ability of insulin to inhibit AMPK, other downstream signaling pathways of insulin are intact, because insulin activation of glycolysis, glucose oxidation, and phosphorylation of Ser473 of Akt (Figures 1 and 2 and supplemental Figure IIA) was not affected by the presence of fatty acid. This suggests that, in mouse heart, there is a fatty acid–dependent insulin-signaling pathway that modifies AMPK activity and a fatty acid independent insulin-signaling pathway that modifies myocardial metabolism.
These observations are of particular importance, as it has been shown that insulin can inhibit the ischemia-induced activation of AMPK,34 which would benefit the ischemic and reperfused myocardium by decreasing fatty acid oxidation. However, these previous studies were performed in the absence of fatty acids. It has also been shown that during reperfusion of ischemic hearts oxidative metabolism quickly recovers, but high rates of fatty acid oxidation result in low glucose oxidation rates attributable to the Randle cycle.24,30,31,35 This reduction in glucose oxidation is detrimental during reperfusion as it contributes to the severity of acidosis in the ischemic and reperfused heart. Ischemia-induced activation of AMPK contributes to the low glucose oxidation rates during reperfusion of ischemic hearts, secondary to stimulation of fatty acid oxidation.10 Insulin may attenuate these effects by inhibiting AMPK. However, we observed that insulin only inhibited AMPK activity in hearts perfused in the absence of fatty acids. Although insulin only induces a 15% inhibition in AMPK activity at the end of ischemia, we may be missing a larger effect as Beauloye et al showed that ischemia-induced AMPK activation peaks after 10 minutes of global ischemia and decreases with extended ischemic times.34 Therefore, insulin may suppress AMPK activation to a greater extent at 10 minutes of ischemia where peak activation occurs.
The cardioprotective effect of insulin has been well established in experimental studies.5–7 It has been suggested that this effect is attributable to either a positive inotropic effect or to a metabolic effect of insulin, such as the stimulation of anaerobic glycolysis to maintain basal ATP production and to switch the preferred oxidative substrate to glucose, which is a more oxygen-efficient substrate and does not produce the toxic intermediates of fatty acid oxidation. As seen in Figure 3, we have confirmed that insulin produces a cardioprotective effect in the absence of fatty acids, improving recovery of cardiac power by 47%. The beneficial effect of insulin during ischemia and reperfusion in the absence of fatty acids may be attributable to an acceleration of glycolysis, which would increase the amount of ATP being produced by the heart. In addition, proton production from glucose metabolism was not significantly elevated by insulin in hearts perfused in the absence of fatty acids. Therefore, the beneficial effects of insulin would be 2-fold, by increasing ATP production by the stimulation of glycolysis and glucose oxidation.
A surprising finding from this study is that in the presence of high fat, insulin impaired the recovery of mechanical function during reperfusion. Like the no-fat condition, insulin stimulated both glycolysis and glucose oxidation during the aerobic period and the reperfusion period. However, because insulin produced a greater stimulation of glycolysis than glucose oxidation, there was an increased in proton production from glucose metabolism during reperfusion (Figure 5). This may explain the detrimental effects of insulin during high-fat perfusion.30,31 As rates of fatty acid oxidation increase, this increases intracellular concentrations of acetyl–coenzyme A (acetyl-CoA), which is an allosteric inhibitor of pyruvate dehydrogenase (PDH). Indeed, the failure of insulin to inhibit AMPK activity may also contribute to this phenomenon. AMPK activation may lead to an acceleration of glucose uptake, thus supplying substrate for glycolysis, but also leads to phosphorylation of acetyl-CoA carboxylase, leading to a decrease in malonyl-CoA levels and an acceleration of fatty acid oxidation, which suppresses glucose oxidation by the abovementioned mechanism (supplemental Table II).
However, proton production during reperfusion in the high-fat plus insulin group was not increased significantly from values in the no-fat plus insulin group, despite dramatic differences in the effect on insulin on postischemic contractile function. The benefits of insulin on recovery of contractile function in the no-fat hearts may be related to an increase in energy supply to these hearts. The no-fat group may be energy starved and energetically compromised; thus, energy supply would be a better determinant of postischemic contractile function than proton production. Insulin decreased the AMP/ATP ratio during aerobic perfusion and increased ATP at the end of reperfusion in the no-fat group (supplemental Table III). However, in the presence of high fat, insulin did not change the AMP/ATP ratio during either aerobic perfusion or during reperfusion.
During the preparation of this report, a study by Horman et al was published suggesting that insulin may inhibit AMPK via Akt phosphorylation of Ser485/491 on the subunit of AMPK, corresponding with a previous report that Akt activity negatively correlates with AMPK Thr172 phosphorylation.36,37 Contradictory results were obtained in the present study. The addition of insulin produced an increase in phosphorylation of Akt at Ser473, which is indicative of Akt activity. Despite this apparent activation of this insulin-signaling pathway, there was no downstream inhibition of AMPK in the no fat palmitate group. This difference may be attributable to the use of a supraphysiological insulin concentration of 100 nmol · L–1 in the Horman study, compared with a more physiological concentration (0.6 nmol · L–1) in the present study. The only clinical study to report arterial insulin concentrations after GIK administration was a study of coronary surgery patients with type 2 diabetes. High-dose GIK therapy increased insulin concentrations to 10.3 nmol · L–1 following administration.38
Considerable interest has also focused on GIK in both AMI and cardiac surgery patients.3,4 However, the recent large CREATE trial did not show GIK effectiveness. It is possible that the discrepant results may be related to differences in plasma fatty acid levels in the patient population. Despite the fact that some of the benefits of GIK have been attributed to a lowering of plasma free fatty acids, previous trials have not determined what effect GIK has on plasma fatty acids levels post–myocardial infarction or postsurgery. In addition, the time of administration of GIK may play a key role in its efficacy, as the only group in the CREATE-ECLA trial with a trend to improvement was the population that received GIK before percutaneous coronary intervention, and this did reach statistical significance in a more recent trial.4,39
In summary, this study demonstrates that the cardioprotective effect of insulin and its ability to modify AMPK activity in the isolated ejecting mouse heart is highly dependent on the concentration of fatty acids present in the perfusate. However, other insulin-signaling pathways, such as modification of glucose metabolism, are still intact in the presence of clinically relevant high concentrations of fatty acids. These findings have important implications in the use of insulin therapy for the treatment of ischemic heart disease.
Acknowledgments
Sources of Funding
Funded by a grant from the Canadian Institutes of Health Research. C.D.L.F. holds an Alberta Heritage for Medical Research (AHFMR) Studentship and a Natural Sciences and Engineering Research Council Scholarship. G.D.L. is an AHFMR Medical Scientist.
Disclosures
None.
Footnotes
Original received December 17, 2005; revision received May 10, 2006; accepted May 19, 2006.
References
Sodi-Pallares D, Ponce de LJ, Bisteni A, Medrano GA. Potassium, glucose, and insulin in myocardial infarction. Lancet. 1969; 1: 1315–1316. [Order article via Infotrieve]
Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction: an overview of randomized placebo-controlled trials. Circulation. 1997; 96: 1152–1156.
Diaz R, Paolasso EA, Piegas LS, Tajer CD, Moreno MG, Corvalan R, Isea JE, Romero G. Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) Collaborative Group. Circulation. 1998; 98: 2227–2234.
Mehta SR, Yusuf S, Diaz R, Zhu J, Pais P, Xavier D, Paolasso E, Ahmed R, Xie C, Kazmi K, Tai J, Orlandini A, Pogue J, Liu L. Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial. JAMA. 2005; 293: 437–446.
Doenst T, Richwine RT, Bray MS, Goodwin GW, Frazier OH, Taegtmeyer H. Insulin improves functional and metabolic recovery of reperfused working rat heart. Ann Thorac Surg. 1999; 67: 1682–1688.
Zaha V, Francischetti I, Doenst T. Insulin improves postischemic recovery of function through PI3K in isolated working rat heart. Mol Cell Biochem. 2003; 247: 229–232. [Order article via Infotrieve]
Fischer-Rasokat U, Beyersdorf F, Doenst T. Insulin addition after ischemia improves recovery of function equal to ischemic preconditioning in rat heart. Basic Res Cardiol. 2003; 98: 329–336. [Order article via Infotrieve]
Winder WW, Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol. 1999; 277: E1–E10.
Hardie DG, Hawley SA. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays. 2001; 23: 1112–1119. [Order article via Infotrieve]
Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. 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. 1995; 270: 17513–17520.
Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, Lopaschuk GD. 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. 1996; 1301: 67–75. [Order article via Infotrieve]
Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den BG, Carling D, Hue L. Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr Biol. 2000; 10: 1247–1255. [Order article via Infotrieve]
Russell RR III, Bergeron R, Shulman GI, Young LH. Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol. 1999; 277: H643–H649.
Kantor PF, Dyck JR, Lopaschuk GD. Fatty acid oxidation in the reperfused ischemic heart. Am J Med Sci. 1999; 318: 3–14. [Order article via Infotrieve]
Lopaschuk GD, Belke DD, Gamble J, Itoi T, Schonekess BO. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta. 1994; 1213: 263–276. [Order article via Infotrieve]
Liedtke AJ, Demaison L, Eggleston AM, Cohen LM, Nellis SH. Changes in substrate metabolism and effects of excess fatty acids in reperfused myocardium. Circ Res. 1988; 62: 535–542.
Lerch R, Tamm C, Papageorgiou I, Benzi RH. Myocardial fatty acid oxidation during ischemia and reperfusion. Mol Cell Biochem. 1992; 116: 103–109. [Order article via Infotrieve]
Lopaschuk GD, Spafford MA, Davies NJ, Wall SR. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res. 1990; 66: 546–553.
Neely JR, Morgan HE. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annu Rev Physiol. 1974; 36: 413–459.
Dennis SC, Gevers W, Opie LH. Protons in ischemia: where do they come from; where do they go to J Mol Cell Cardiol. 1991; 23: 1077–1086. [Order article via Infotrieve]
Karmazyn M, Moffat MP. Role of Na+/H+ exchange in cardiac physiology and pathophysiology: mediation of myocardial reperfusion injury by the pH paradox. Cardiovasc Res. 1993; 27: 915–924. [Order article via Infotrieve]
Scholz W, Albus U. Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion. Basic Res Cardiol. 1993; 88: 443–455. [Order article via Infotrieve]
Orchard CH, Houser SR, Kort AA, Bahinski A, Capogrossi MC, Lakatta EG. Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium. J Gen Physiol. 1987; 90: 145–165.
Lopaschuk GD, Wambolt RB, Barr RL. An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. J Pharmacol Exp Ther. 1993; 264: 135–144.
Opie LH. Myocardial ischemia-metabolic pathways and implications of increased glycolysis. Cardiovasc Drugs Ther. 1990; 4 (suppl 4): 777–790. [Order article via Infotrieve]
Clark H, Carling D, Saggerson D. Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids. Eur J Biochem. 2004; 271: 2215–2224.
Lopaschuk GD, Collins-Nakai R, Olley PM, Montague TJ, McNeil G, Gayle M, Penkoske P, Finegan BA. Plasma fatty acid levels in infants and adults after myocardial ischemia. Am Heart J. 1994; 128: 61–67. [Order article via Infotrieve]
Larsen TS, Belke DD, Sas R, Giles WR, Severson DL, Lopaschuk GD, Tyberg JV. The isolated working mouse heart: methodological considerations. Pflugers Arch. 1999; 437: 979–985. [Order article via Infotrieve]
Barr RL, Lopaschuk GD. Direct measurement of energy metabolism in the isolated working rat heart. J Pharmacol Toxicol Methods. 1997; 38: 11–17. [Order article via Infotrieve]
Liu B, Clanachan AS, Schulz R, Lopaschuk GD. Cardiac efficiency is improved after ischemia by altering both the source and fate of protons. Circ Res. 1996; 79: 940–948.
Liu Q, Docherty JC, Rendell JC, Clanachan AS, Lopaschuk GD. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J Am Coll Cardiol. 2002; 39: 718–725.
Gamble J, Lopaschuk GD. Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism. 1997; 46: 1270–1274. [Order article via Infotrieve]
Makinde AO, Gamble J, Lopaschuk GD. Upregulation of 5'-AMP-activated protein kinase is responsible for the increase in myocardial fatty acid oxidation rates following birth in the newborn rabbit. Circ Res. 1997; 80: 482–489.
Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, Hue L. Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett. 2001; 505: 348–352. [Order article via Infotrieve]
Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963; 1: 785–789. [Order article via Infotrieve]
Horman S, Vertommen D, Heath R, Neumann D, Mouton V, Woods A, Schlattner U, Wallimann T, Carling D, Hue L, Rider MH. Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase alpha-subunits in heart via hierarchical phosphorylation of Ser485/491. J Biol Chem. 2006; 281: 5335–5340.
Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JRB. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem. 2003; 278: 39422–39427.
Szabo Z, Arnqvist H, Hakanson E, Jorfeldt L, Svedjeholm R. Effects of high-dose glucose-insulin-potassium on myocardial metabolism after coronary surgery in patients with Type II diabetes. Clin Sci (Lond). 2001; 101: 37–43. [Order article via Infotrieve]
Yazici M, Demircan S, Durna K, Yasar E, Acar Z, Sahin M. Effect of glucose-insulin-potassium infusion on myocardial damage due to percutaneous coronary revascularization. Am J Cardiol. 2005; 96: 1517–1520. [Order article via Infotrieve]
您现在查看是摘要介绍页，详见4,29附件。