Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation

Objective

The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization.

Methods

Isolated rat hearts were perfused with glucosamine (0–10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by ¹³C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36.

Results

Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation.

Conclusion/Interpretation

These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.     

Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations

Because of the importance of long-chain fatty acids (LCFAs) as a myocardial energy substrate, myocardial LCFA metabolism has been of particular interest for the understanding of cardiac pathophysiology. Recently, by using radiolabeled LCFA analogues, myocardial LCFA metabolism has been clinically evaluated, which revealed a total defect of myocardial LCFA accumulation in a small number of subjects. The mechanism for the cellular LCFA uptake process is still disputable, but recent results suggest that fatty acid translocase (FAT)/CD36 is a transporter in the heart. In the present study, we analyzed mutations and protein expression of the FAT/CD36 gene in 47 patients who showed total lack of the accumulation of a radiolabeled LCFA analogue in the heart. All the patients carried two mutations in the FAT/CD36 gene, and expression of the FAT/CD36 protein was not detected on either platelet or monocyte membranes. Our results showed the link between mutations of the FAT/CD36 gene and a defect in the accumulation of LCFAs in the human heart.     

Calmodulin kinase II inhibition enhances ischemic preconditioning by augmenting ATP-sensitive K+ current

Mice with genetic inhibition (AC3-I) of the multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaMKII) have improved cardiomyocyte survival after ischemia. Some K(+) currents are up-regulated in AC3-I hearts, but it is unknown if CaMKII inhibition increases the ATP sensitive K(+) current (I(KATP)) that underlies ischemic preconditioning (IP) and confers resistance to ischemia. We hypothesized increased I(KATP) was part of the mechanism for improved ventricular myocyte survival during ischemia in AC3-I mice. AC3-I hearts were protected against global ischemia due to enhanced IP compared to wild type (WT) and transgenic control (AC3-C) hearts. IKATP was significantly increased, while the negative regulatory dose-dependence of ATP was unchanged in AC3-I compared to WT and AC3-C ventricular myocytes, suggesting that CaMKII inhibition increased the number of functional I(KATP) channels available for IP. We measured increased sarcolemmal Kir6.2, a pore-forming I(KATP) subunit, but not a change in total Kir6.2 in cell lysates or single channel I(KATP) opening probability from AC3-I compared to WT and AC3-C ventricles, showing CaMKII inhibition increased sarcolemmal I(KATP) channel expression. There were no differences in mRNA for genes encoding I(KATP) channel subunits in AC3-I, WT and AC3-C ventricles. The I(KATP) opener pinacidil (100 microM) reduced MI area in WT to match AC3-I hearts, while the I(KATP) antagonist HMR1098 (30 microM) increased MI area to an equivalent level in all groups, indicating that increased I(KATP) and augmented IP are important for reduced ischemic cell death in AC3-I hearts. Our study results show CaMKII inhibition enhances beneficial effects of IP by increasing I(KATP).     

Hypoxia-induced fatty acid transporter translocation increases fatty acid transport and contributes to lipid accumulation in the heart

Protein-mediated LCFA transport across plasma membranes is highly regulated by the fatty acid transporters FAT/CD36 and FABPpm. Physiologic stimuli (insulin stimulation, AMP kinase activation) induce the translocation of one or both transporters to the plasma membrane and increase the rate of LCFA transport. In the hypoxic/ischemic heart, intramyocardial lipid accumulation has been attributed to a reduced rate of fatty acid oxidation. However, since acute hypoxia (15 min) activates AMPK, we examined whether an increased accumulation of intramyocardial lipid during hypoxia was also attributable to an increased rate of LCFA uptake as a result AMPK-induced translocation of FAT/CD36 and FABPpm. In cardiac myocytes, hypoxia (15 min) induced the redistribution of FAT/CD36 from an intracellular pool (LDM) (-25%, P<0.05) to the plasma membranes (PM) (+54%, P<0.05). Hypoxia also induced an increase in FABPpm at the PM (+56%, P<0.05) and a concomitant FABPpm reduction in the LDM (-24%, P<0.05). Similarly, in intact, Langendorff perfused hearts, hypoxia induced the translocation of a both FAT/CD36 and FABPpm to the PM (+66% and +61%, respectively, P<0.05), with a concomitant decline in FAT/CD36 and FABPpm in the LDM (-24% and -23%, respectively, P<0.05). Importantly, the increased plasmalemmal content of these transporters was associated with increases in the initial rates of palmitate uptake into cardiac myocytes (+40%, P<0.05). Acute hypoxia also redirected palmitate into intracellular lipid pools, mainly to PL and TG (+48% and +28%, respectively, P<0.05), while fatty acid oxidation was reduced (-35%, P<0.05). Thus, our data indicate that the increased intracellular lipid accumulation in hypoxic hearts is attributable to both: (a) a reduced rate of fatty acid oxidation and (b) an increased rate of fatty acid transport into the heart, the latter being attributable to a hypoxia-induced translocation of fatty acid transporters.     

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent

The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.     

Myocardial ischemia differentially regulates LKB1 and an alternate 5'-AMP-activated protein kinase kinase

During myocardial ischemia, activation of 5'-AMP-activated protein kinase (AMPK) leads to the stimulation of glycolysis and fatty acid oxidation. Together these metabolic changes contribute to cardiac dysfunction. Although AMPK signaling in the ischemic heart is well characterized, the relative contribution of phosphorylation by AMPK kinase (AMPKK), and positive allosterism by the ratios of AMP:ATP and creatine (Cr):phosphocreatine (PCr), in stimulating AMPK during ischemia are unknown. In hearts subjected to severe ischemia, the ratios of AMP:ATP and Cr:PCr were significantly elevated as compared with aerobic hearts. Severe ischemia stimulated AMPK signaling, as demonstrated by an increase in both AMPK activity and acetyl-CoA carboxylase phosphorylation. Although AMPK phosphorylation was increased by severe ischemia, the protein abundance and activity of the recently identified AMPKK, LKB1, were similar between aerobic and severely ischemic hearts. However, in contrast to LKB1, the activity of AMPKK was stimulated in severely ischemic hearts. To further delineate the relative roles of positive allosterism and AMPKK in the regulation of AMPK during ischemia, hearts were subjected to mild ischemia. Although mild ischemia did not alter the ratios of AMP:ATP and Cr:PCr, mild ischemia increased AMPK activity and increased AMPK phosphorylation. Mild ischemia also stimulated the activity of AMPKK. In summary, we demonstrate that myocardial ischemia stimulates AMPK via an AMPKK other than LKB1. Additionally, we show that changes in high energy phosphates are not essential for the activation of AMPK by ischemia. Our data emphasize the critical role AMPKK plays in mediating AMPK signaling during myocardial ischemia.     

AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart

Myocardial ischemia is the leading cause of all cardiovascular deaths in North America. Myocardial ischemia is accompanied by profound changes in metabolism including alterations in glucose and fatty acid metabolism, increased uncoupling of glucose oxidation from glycolysis and accumulation of protons within the myocardium. These changes can contribute to a poor functional recovery of the heart. One key player in the ischemia-induced alteration in fatty acid and glucose metabolism is 5'AMP-activated protein kinase (AMPK). Accumulating evidence suggest that activation of AMPK during myocardial ischemia both increases glucose uptake and glycolysis while also increasing fatty acid oxidation during reperfusion. Gain-of-function mutations of AMPK in cardiac muscle may also be causally related to the development of hypertrophic cardiomyopathies. Therefore, a better understanding of role of AMPK in cardiac metabolism is necessary to appropriately modulate its activity as a potential therapeutic target in treating ischemia reperfusion injuries. This review attempts to update some of the recent findings that delineate various pathways through which AMPK regulates glucose and fatty acid metabolism in the ischemic myocardium.     

Electrostimulation enhances FAT/CD36-mediated long-chain fatty acid uptake by isolated rat cardiac myocytes

We investigated palmitate uptake and utilization by contracting cardiac myocytes in suspension to explore the link between long-chain fatty acid (FA) uptake and cellular metabolism, in particular the role of fatty acid translocase (FAT)/CD36-mediated transsarcolemmal FA transport. For this, an experimental setup was developed to electrically stimulate cardiomyocytes in multiple parallel incubations. Electrostimulation at voltages > or =170 V resulted in cellular contraction with no detrimental effect on cellular integrity. At 200 V and 4 Hz, palmitate uptake (measured after 3-min incubation) was enhanced 1.5-fold. In both quiescent and contracting myocytes, after their uptake, palmitate was largely and rapidly esterified, mainly into triacylglycerols. Palmitate oxidation (measured after 30 min) contributed to 22% of palmitate taken up by quiescent cardiomyocytes and, after stimulation at 4 Hz, was increased 2.8-fold to contribute to 39% of palmitate utilization. The electrostimulation-mediated increase in palmitate uptake was blocked in the presence of either verapamil, a contraction inhibitor, or sulfo-N-succinimidyl-FA esters, specific inhibitors of FAT/CD36. These data indicate that, in contracting cardiac myocytes, palmitate uptake is increased due to increased flux through FAT/CD36.     

CD36 actions in the heart: Lipids,calcium, inflammation,repair and more?

CD36 is a multifunctional immuno-metabolic receptor with many ligands. One of its physiological functions in the heart is the high-affinity uptake of long-chain fatty acids (FAs) from albumin and triglyceride rich lipoproteins. CD36 deletion markedly reduces myocardial FA uptake in rodents and humans. The protein is expressed on endothelial cells and cardiomyocytes and at both sites is likely to contribute to FA uptake by the myocardium. CD36 also transduces intracellular signaling events that influence how the FA is utilized and mediate metabolic effects of FA in the heart. CD36 transduced signaling regulates AMPK activation in a way that adjusts oxidation to FA uptake. It also impacts remodeling of myocardial phospholipids and eicosanoid production, effects exerted via influencing intracellular calcium (iCa^2 +) and the activation of phospholipases. Under excessive FA supply CD36 contributes to lipid accumulation, inflammation and dysfunction. However, it is also important for myocardial repair after injury via its contribution to immune cell clearance of apoptotic cells. This review describes recent progress regarding the multiple actions of CD36 in the heart and highlights those areas requiring future investigation. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

The role of CD36 in the regulation of myocardial lipid metabolism

Since the heart has one of the highest energy requirements of all organs in the body, it requires a constant and plentiful supply of fuel to function properly. Mitochondrial oxidation of lipids provides a major source of ATP for the heart, and the cellular processes that regulate lipid uptake and utilization are important contributors to maintaining proper myocardial energetic status. Although numerous proteins are coordinately regulated in order to ensure proper fatty acid utilization in the cardiomyocyte, a key first step in this process is the entry of fatty acids into the cell. An important protein involved in the transport of fatty acids into the cardiomyocyte is the plasma membrane-associated protein known as fatty acid translocase (FAT; also known as CD36). While multiple proteins are involved in facilitating fatty acid uptake in the heart, CD36 accounts for approximately 50–70% of the total fatty acid taken up in cardiomyocytes. As such, myocardial metabolism of fatty acids may depend upon proper CD36 function. Consistent with this, changes in CD36 levels/function have been implicated in the alteration of myocardial metabolism in the pathophysiology of certain cardiovascular diseases. As such, a better understanding of the role and function of CD36 in the heart may provide important insights for the development of new treatments for specific cardiovascular diseases. Herein, we review the role of CD36 in myocardial lipid metabolism in the healthy heart and describe how CD36-mediated alterations in lipid metabolism may contribute to cardiovascular disease. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

Dynamics of Ca(2+)/calmodulin-dependent protein kinase II following acute myocardial ischemia-translocation and autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase (CaMK) family is responsive to changes in the intracellular Ca(2+) concentration. However, their functions have not been well established in the ischemia/reperfusion heart. The effects of myocardial ischemia on CaMKII, the most strongly expressed form, were investigated using isolated rat hearts. Rat hearts were rendered globally ischemic by stopping perfusion for 15 min, and then reperfused, heart ventricles being analyzed in each phase. Western blotting detected a decrease in the cytosolic and concomitant increase in the particulate fraction of CaMKII following transient ischemia. Redistribution to the cytosol was revealed on reperfusion. Northern blot showed CaMKII gene expression decreased by ischemia. Furthermore, autoradiography and confocal immunohistochemical findings provided autophosphorylation of CaMKII in the cytosol, ischemia causing decrease, with gradual recovery on reperfusion. These results indicate a transient partial translocation of CaMKII accompanied by kinase activity, with residual myocardial CaMKII undergoing autophosphorylation during ischemia and reperfusion, demonstrating two different characteristic dynamics of CaMKII.     

Effects of adenosine on myocardial glucose and palmitate metabolism after transient ischemia: role of 5'-AMP-activated protein kinase

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.     

Targeting fatty acid and carbohydrate oxidation--a novel therapeutic intervention in the ischemic and failing heart

Cardiac ischemia and its consequences including heart failure, which itself has emerged as the leading cause of morbidity and mortality in developed countries are accompanied by complex alterations in myocardial energy substrate metabolism. In contrast to the normal heart, where fatty acid and glucose metabolism are tightly regulated, the dynamic relationship between fatty acid β-oxidation and glucose oxidation is perturbed in ischemic and ischemic-reperfused hearts, as well as in the failing heart. These metabolic alterations negatively impact both cardiac efficiency and function. Specifically there is an increased reliance on glycolysis during ischemia and fatty acid β-oxidation during reperfusion following ischemia as sources of adenosine triphosphate (ATP) production. Depending on the severity of heart failure, the contribution of overall myocardial oxidative metabolism (fatty acid β-oxidation and glucose oxidation) to adenosine triphosphate production can be depressed, while that of glycolysis can be increased. Nonetheless, the balance between fatty acid β-oxidation and glucose oxidation is amenable to pharmacological intervention at multiple levels of each metabolic pathway. This review will focus on the pathways of cardiac fatty acid and glucose metabolism, and the metabolic phenotypes of ischemic and ischemic/reperfused hearts, as well as the metabolic phenotype of the failing heart. Furthermore, as energy substrate metabolism has emerged as a novel therapeutic intervention in these cardiac pathologies, this review will describe the mechanistic bases and rationale for the use of pharmacological agents that modify energy substrate metabolism to improve cardiac function in the ischemic and failing heart. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Targeting fatty acid and carbohydrate oxidation — A novel therapeutic intervention in the ischemic and failing heart

Cardiac ischemia and its consequences including heart failure, which itself has emerged as the leading cause of morbidity and mortality in developed countries are accompanied by complex alterations in myocardial energy substrate metabolism. In contrast to the normal heart, where fatty acid and glucose metabolism are tightly regulated, the dynamic relationship between fatty acid β-oxidation and glucose oxidation is perturbed in ischemic and ischemic-reperfused hearts, as well as in the failing heart. These metabolic alterations negatively impact both cardiac efficiency and function. Specifically there is an increased reliance on glycolysis during ischemia and fatty acid β-oxidation during reperfusion following ischemia as sources of adenosine triphosphate (ATP) production. Depending on the severity of heart failure, the contribution of overall myocardial oxidative metabolism (fatty acid β-oxidation and glucose oxidation) to adenosine triphosphate production can be depressed, while that of glycolysis can be increased. Nonetheless, the balance between fatty acid β-oxidation and glucose oxidation is amenable to pharmacological intervention at multiple levels of each metabolic pathway. This review will focus on the pathways of cardiac fatty acid and glucose metabolism, and the metabolic phenotypes of ischemic and ischemic/reperfused hearts, as well as the metabolic phenotype of the failing heart. Furthermore, as energy substrate metabolism has emerged as a novel therapeutic intervention in these cardiac pathologies, this review will describe the mechanistic bases and rationale for the use of pharmacological agents that modify energy substrate metabolism to improve cardiac function in the ischemic and failing heart. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Regulation of cardiac malonyl-CoA content and fatty acid oxidation during increased cardiac power

Myocardial fatty acid oxidation is regulated by carnitine palmitoyltransferase I (CPT I), which is inhibited by malonyl-CoA. Increased cardiac power causes a fall in malonyl-CoA content and accelerated fatty acid oxidation; however, the mechanism for the decrease in malonyl-CoA is unclear. Malonyl-CoA is formed by acetyl-CoA carboxylase (ACC) and degraded by malonyl-CoA decarboxylase (MCD); thus a fall in malonyl-CoA could be due to activation of MCD, inhibition of ACC, or both. This study assessed the effects of increased cardiac power on malonyl-CoA content and ACC and MCD activities. Anesthetized pigs were studied under control conditions and during increased cardiac power in response to dobutamine infusion and aortic constriction alone, under hyperglycemic conditions, or with the CPT I inhibitor oxfenicine. An increase in cardiac power was accompanied by increased myocardial O(2) consumption, decreased malonyl-CoA concentration, and increased fatty acid oxidation. There were no differences among groups in activity of ACC or AMP-activated protein kinase (AMPK), which physiologically inhibits ACC. There also were no differences in V(max) or K(m) of MCD. Previous studies have demonstrated that AMPK can be inhibited by protein kinase B (PKB); however, PKB was activated by dobutamine and the elevated insulin that accompanied hyperglycemia, but there was no effect on AMPK activity. In conclusion, the fall in malonyl-CoA and increase in fatty acid oxidation that occur with increased cardiac work were not due to inhibition of ACC or activation of MCD, suggesting alternative regulatory mechanisms for the work-induced decrease in malonyl-CoA concentration.     

Apelin-13 increases myocardial progenitor cells and improves repair postmyocardial infarction

Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against myocardial ischemia-reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac functional recovery postmyocardial infarction (post-MI). The present study investigated whether apelin affects PC homing to the infarcted myocardium, thereby mediating repair and functional recovery post-MI. Mice were infarcted by coronary artery ligation, and apelin-13 (1 mg·kg(-1)·day(-1)) was injected for 3 days before MI and for 14 days post-MI. Homing of vascular PCs [CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/stromal cell-derived factor (SDF)-1α(+), and CD133(+)/CXC chemokine receptor (CXCR)-4(+)] into the ischemic area was examined. Myocardial Akt, endothelial nitric oxide synthase (eNOS), VEGF, jagged1, notch3, SDF-1α, and CXCR-4 expression were assessed at 24 h and 14 days post-MI. Functional analyses were performed on day 14 post-MI. Mice that received apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR-4 expression and dramatically increased the number of CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/SDF-1α(+), and c-Kit(+)/CXCR-4(+) cells in infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, jagged1, and notch3 expression in ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Furthermore, treatment with apelin-13 promoted myocardial angiogenesis and attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI. Apelin-13 increases angiogenesis and improves cardiac repair post-MI by a mechanism involving the upregulation of SDF-1α/CXCR-4 and homing of vascular PCs.     

Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis

Leptin regulates fatty acid metabolism in liver, skeletal muscle, and pancreas by partitioning fatty acids into oxidation rather than triacylglycerol (TG) storage. Although leptin receptors are present in the heart, it is not known whether leptin also regulates cardiac fatty acid metabolism. To determine whether leptin directly regulates cardiac fatty acid metabolism, isolated working rat hearts were perfused with 0.8 mm [9,10-(3)H]palmitate and 5 mm [1-(14)C]glucose to measure palmitate and glucose oxidation rates. Leptin (60 ng/ml) significantly increased palmitate oxidation rates 60% above control hearts (p < 0.05) and decreased TG content by 33% (p < 0.05) over the 60-min perfusion period. In contrast, there was no difference in glucose oxidation rates between leptin-treated and control hearts. Although leptin did not affect cardiac work, oxygen consumption increased by 30% (p < 0.05) and cardiac efficiency was decreased by 42% (p < 0.05). AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels. Leptin has also been shown to increase fatty acid oxidation in skeletal muscle through the activation of AMPK. However, we demonstrate that leptin had no significant effect on AMPK activity, AMPK phosphorylation state, ACC activity, or malonyl-CoA levels. AMPK activity and its phosphorylation state were also unaffected after 5 and 10 min of perfusion in the presence of leptin. The addition of insulin (100 microunits/ml) to the perfusate reduced the ability of leptin to increase fatty acid oxidation and decrease cardiac TG content. These data demonstrate for the first time that leptin activates fatty acid oxidation and decreases TG content in the heart. We also show that the effects of leptin in the heart are independent of changes in the AMPK-ACC-malonyl-CoA axis.