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Activation of 5'-AMP-activated protein kinase (AMPK) may benefit the heart during ischemia-reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair the functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative (DN) [[alpha].sub.2]-subunit of AMPK (AMPK-[[alpha].sub.2] DN) to 20 min of global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-[sup.14]C]glucose, 0.4 mM [9, 10-[sup.3]H]palmitate, and 100 [micro]U/ml insulin. AMPK-[[alpha].sub.2] DN hearts had reduced AMPK activity at the end of reperfusion (82 [+ or -] vs. 141 [+ or -] 7 pmol x [mg.sup.-1] x [min.sup.-1]) with no changes in high-energy phosphates. Despite this, AMPK-[[alpha].sub.2] DN hearts had improved recovery of function during reperfusion (14.9 [+ or -] 0.8 vs. 9.4 [+ or -] 1.4 beats x [min.sup.-1] x mmHg x [10.sup.-3]). During reperfusion, fatty acid oxidation provided 44.0 [+ or -] 2.8% of total acetyl-CoA in AMPK-[[alpha].sub.2] DN hearts compared with 55.0 [+ or -] 3.2% in control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4 [+ or -] 9.4% vs. 85.3 [+ or -] 4.3%). These data demonstrate that the suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function after ischemia. ischemia-reperfusion; fatty acid oxidation; glycolysis; glucose oxidation

The role of proton ([H.sup.+]) production from glucose metabolism in the recovery of myocardial function during postischemic reperfusion and its alteration by insulin and other metabolic modulators were examined. Rat hearts were perfused in vitro with Krebs-Henseleit solution containing palmitate (1.2 retool/l) and glucose (11 retool/l) under nonischemic conditions or during reperfusion following no-flow ischemia. Perfusate contained normal insulin (n-Ins, 50 mU/l), zero insulin (0-1ns), or supplemental insulin (s-Ins, 1,000 mU/l) or other metabolic modulators [dichloroacetate (DCA) at 3 retool/l, oxfenicine at 1 retool/l, and [N.sup.6]-cyclohexyladenosine (CHA) at 0.5 [micro]mol/1]. Relative to n-Ins, 0-Ins depressed rates of glycolysis and glucose oxidation in nonischemic hearts and impaired recovery of postischemic function. Relative to n-lns, s-Ins did not affect aerobic glucose metabolism and did not improve recovery when present during reperfusion. When present during ischemia and reperfusion, s-Ins impaired recovery. Combinations of metabolic modulators with s-Ins stimulated glucose oxidation ~2.5-fold in nonischemic hearts and reduced [H.sup.+] production. DCA and CHA, in combination with s-Ins, improved recovery of function, but addition of oxfenicine to this combination provided no further benefit. Although DCA and CHA were each partially protective in hearts perfused with n-Ins, optimal protection was achieved with DCA + CHA; recovery of function was inversely proportional to [H.sup.+] production during reperfusion. Although supplemental insulin is not beneficial, elimination of [H.sup.+] production from glucose metabolism by simultaneous inhibition of glycolysis and stimulation of glucose oxidation optimizes recovery of postischemic mechanical function. proton production; ischemia-reperfusion; contractile function; rat hearts

Alterations in myocardial glucose metabolism are a key determinant of ischemia-induced depression of left ventricular mechanical function. Since myocardial glycogen is an important source of endogenous glucose, we compared the effects of ischemia on glucose uptake and utilization in isolated working rat hearts in which glycogen content was either replete (G replete, 114 [micro]mol/g dry wt) or partially depleted (G depleted, 71 [micro]mol/g dry wt). The effects of low-flow ischemia (LFI, 0.5 ml/min) on glucose uptake, glycogen turnover (glycogenolysis and glycogen synthesis), glycolysis, adenosine 5'-monophosphate-activated protein kinase (AMPK) activity, and GLUT4 translocation were measured. Relative to preischemic values, LFI caused a time-dependent reduction in glycogen content in both G-replete and G-depleted groups due to an acceleration of glycogenolysis (by 12-fold and 6-fold, respectively). In G-replete hearts, LFI (15 min) decreased glucose uptake (by 59%) and did not affect GLUT4 translocation. In G-depleted hearts, LFI also decreased initially glucose uptake (by 90%) and glycogen synthesis, but after 15 min, when glycogenolysis slowed due to exhaustion of glycogen content, glucose uptake increased (by 31%) in association with an increase in GLUT4 translocation. After 60 min of LFI, glucose uptake, glycogenolysis, and glycolysis recovered to near-preischemic values in both groups. LFI increased AMPK activity in a time-dependent manner in both groups (by 6-fold and 4-fold, respectively). Thus, when glycogen stores are replete before ischemia, ischemia-induced AMPK activation is not sufficient to increase glucose uptake. Under these conditions, an acceleration of glycogen degradation provides sufficient endogenous substrate for glycolysis during ischemia. low-flow ischemia; glucose uptake; glycogen metabolism; glucose metabolism; adenosine-5'-monophosphate-activated protein kinase

p38 mitugen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [[sup.3]H/[sup.14]C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 X 10 rain I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 [micro]M), adenosine (500 [micro]M), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism ([micro]mol*g dry [wt.sup.-1]*[min.sup.-1), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 [+ or -] 0.01, n = 3 vs. 0.48 [+ or -] 0.10, n = 3, P < 0.05) and AMPK (0.00 [+ or -] 0.00, n = 3 vs. 0.26 [+ or -] 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 [+ or -] 0.40, n = 7 vs. 3.95 [+ or -] 0.65, n = 7, P < 0.05) and [H.sup.+] production (2.12 [+ or -] 0.76, n = 7 vs. 6.96 [+ or -] 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 [+ or -] 0.25, n = 6 vs. 0.27 [+ or -] 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 [+ or -] 0.08, n = 7 vs. 0.30 [+ or -] 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection. p38 mitogen-activated protein kinase; 5'-adenosine monophosphateactivated protein kinase; glucose metabolism

Adenosineinduced acceleration of glycolysis in hearts stressed by transient ischemia is accompanied by suppression of glycogen synthesis and by increases in activity of adenosine 5'-monophosphate-activated protein kinase (AMPK). Because p38 mitogen-activated protein kinase (MAPK) may regulate glucose metabolism and may be activated downstream of AMPK, this study determined the effects of the p38 MAPK inhibitors SB202190 and SB203580 on adenosine-induced alterations in glucose utilization and AMPK activity. Studies were performed in working rat hearts perfused aerobically following stressing by transient ischemia (2 x 10-min ischemia followed by 5-min reperfusion). Phosphorylation of AMPK and p38 MAPK each were increased fourfold by adenosine, and these effects were inhibited by either SB202190 or SB203580. Neither of these inhibitors directly affected AMPK activity. Attenuation of the adenosine-induced increase in AMPK and p38 MAPK phosphorylation by SB202190 and SB203580 occurred independently of any change in tissue ATP-to-AMP ratio and did not alter glucose uptake, but it was accompanied by an increase in glycogen synthesis and glycogen content and by inhibition of glycolysis and proton production. There was a significant inverse correlation between the rate of glycogen synthesis and AMPK activity and between AMPK activity and glycogen content. These data demonstrate that AMPK is likely downstream of p38 MAPK in mediating the effects of adenosine on glucose utilization in hearts stressed by transient ischemia. The ability of p38 MAPK inhibitors to relieve the inhibition of glycogen synthesis and to inhibit glycolysis and proton production suggests that these agents may restore adenosine -induced cardioprotection in stressed hearts. adenosine; adenosine 5'-monophosphate-activated protein kinase; glycogen metabolism

Loss of cardioprotection by adenosine in hearts stressed by transient ischemia may be due to its effects on glucose metabolism. In the absence of transient ischemia, adenosine inhibits glycolysis, whereas it accelerates glycolysis after transient ischemia. Inasmuch as 5'-AMP-activated protein kinase (AMPK) is implicated as a regulator of glucose and fatty acid utilization, this study determined whether a differential alteration of AMPK activity contributes to acceleration of glycolysis by adenosine in hearts stressed by transient ischemia. Studies were performed in working rat hearts perfused aerobically under normal conditions or after transient ischemia (two 10-min periods of ischemia followed by 5 min of reperfusion). LV work was not affected by adenosine. AMPK phosphorylation was not affected by transient ischemia; however, phosphorylation and activity were increased nine-and threefold, respectively, by adenosine in stressed hearts. Phosphorylation of acetyl-CoA carboxylase and rates of palmitate oxidation were unaltered. Glycolysis and calculated proton production were increased 1.8-and 1.7-fold, respectively, in hearts with elevated AMPK activity. Elevated AMPK activity was associated with inhibition of glycogen synthesis and unchanged rates of glucose uptake and glycogenolysis. Phentolamine, an [alpha]-adrenoceptor antagonist, which prevents adenosine-induced activation of glycolysis in stressed hearts, prevented AMPK phosphorylation. These data demonstrate that adenosine-induced activation of AMPK after transient ischemia is not sufficient to alter palmitate oxidation or glucose uptake. Rather, activation of AMPK alters partitioning of glucose away from glycogen synthesis; the increase in glycolysis may in part contribute to loss of adenosine-induced cardioprotection in hearts subjected to transient ischemia. glucose metabolism; glucose uptake; glycogen

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 [micro]U/ml insulin or 100 [micro]U/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor [C.sub.2]-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation. insulin; glucose; ceramide; Akt, metabolism

Glucose labeled with hydrogen and carbon isotopes was used to measure glycogen turnover in isolated rat hearts during periods of aerobic metabolism, low-flow ischemia and reperfusion. It is important to take into account the simultaneous measurement of glycogen synthesis and degradation to accurately assess the rate of glycogen turnover. This method detects glycogen in the rat myocardium during aerobic, ischemic and reperfused conditions.

Experiments were performed to examine the ability of triiodothyronine (t3) treatment to improve the mechanical function and cardiac efficiency during reperfusion of ischemic rat hearts by modulation of glucose metabolism. Isolated working rat hearts were subjected to no-flow ischemia and aerobic reperfusion after being perfused with high levels of fatty acids. Results showed that T3 enhanced the recovery of mechanical function and cardiac performance in the post-ischemic rat heart when H+ production from glucose metabolism was lowered.

The effects of selective intrinsic angiotensin II (ANG II) type 1 receptor (AT1-R) blockage with losartan on proton production from glucose metabolism and recovery of function in isolated working rat hearts perfused with Krebs-Henseleit buffer with palmitate, glucose and insulin, were studied. Results showed that the presence of endogenous ANG II played a role in the recovery under control conditions. Also, the losartan-induced inhibition of mechanical recovery was mediated through myocardial AT1-R antagonism and is reversed by displacement of losartan from its receptor by ANG II.

Cardiac performance was monitored during continuous infusions of ONOO- in isolated working rat hearts to determine the effects of the peroxynitrite on cardiac contraction. Bolus injections of ONOO- in Langendorff-perfused rat hearts impaired cardiac contractile function in a time- and dose-dependent manner without altering myocardial oxygen consumption or adenosine '3,5'-triphosphate (ATP) synthesis. Furthermore, ONOO- reduced the efficiency of the heart to utilize myocardial ATP during cardiac contraction.

Previous studies have demonstrated the cardioprotective effects of adenosine (Ado) during ischemia and reperfusion, but the mechanisms underlying the role of Ado have not been resolved. Herein, experiments on isolated rabbit hearts showed that Ado influences the coupling of glycolysis to glucose oxidation following ischemia and that the effect of Ado was determined by the preischemic glycogen content of the heart.

Mitochondrial tricarboxylic acid (TCA) cycle activity and oxidative phosphorylation are quickly retrieved during the reperfusion of rat hearts subjected to ischemia. The rate of recovery of the mitochondrial metabolism is greater than that of contractile function, and culminates in a rapid drop in cardiac efficiency during reperfusion. The efficiency of coupling between cardiac work and mitochondrial respiration, and cardiac work and TCA cycle activity, decrease during reperfusion. The coupling of TCA cycle activity and myocardial O2 consumption, however, revert to the preishemic values.

Ischemic preconditioning minimizes the harmful effects of prolonged periods of ischemia by inducing short ischemic episodes. These episodes were found to be accompanied by a reduction in glucose metabolism, which eventually reduces the amount of protons available from glycolysis. Rat hearts induced to undergo ischemia were also found to exhibit a higher concentration of adenosine and adenosine triphospate.