Regulation of myocardial fatty acid oxidation by substrate supply

We tested the hypothesis that myocardial substrate supply regulates fatty acid oxidation independent of changes in acetyl-CoA carboxylase (ACC) and 5'-AMP-activated protein kinase (AMPK) activities. Fatty acid oxidation was measured in isolated working rat hearts exposed to different concentrations of exogenous long-chain (0.4 or 1.2 mM palmitate) or medium-chain (0.6 or 2.4 mM octanoate) fatty acids. Fatty acid oxidation was increased with increasing exogenous substrate concentration in both palmitate and octanoate groups. Malonyl-CoA content only rose as acetyl-CoA supply from octanoate oxidation increased. The increases in octanoate oxidation and malonyl-CoA content were independent of changes in ACC and AMPK activity, except that ACC activity increased with very high acetyl-CoA supply levels. Our data suggest that myocardial substrate supply is the primary mechanism responsible for alterations in fatty acid oxidation rates under nonstressful conditions and when substrates are present at physiological concentrations. More extreme variations in substrate supply lead to changes in fatty acid oxidation by the additional involvement of intracellular regulatory pathways.     

Differential effects of acetate on palmitate and octanoate oxidation: Segregation of acetyl CoA pools

In isolated hepatic mitochondria, sodium acetate had little effect on the oxidation of octanoate, but conspicuously inhibited the oxidation of palmitate. This differential effect of acetate on long-chain and short-chain fatty acid oxidation is not due to inhibition of the activation or transfer of long-chain fatty acids into the mitochondria. Both palmitate and octanoate reduced CO₂ production from acetate. Palmitate and octanoate mutually inhibited CO₂ production from each other to the same extent. Acetate stimulated ketogenesis from palmitoyl-1-carnitine to the same extent as it inhibited oxygen uptake and CO₂ production from palmitate. This suggests that acetate causes a redistribution of the end products of palmitate oxidation toward ketogenesis rather than toward total oxidation to CO₂ and H₂O. Acetyl CoA derived from acetate or palmitate may share a common pool or pathway, thus each is mutally inhibitory toward the oxidation of the other. Either because of the existence of separate pools, or because octanoate is the preferred substrate, acetate metabolism does not inhibit O₂ uptake or CO₂ production from octanoate, whereas the oxidation of octanoate dilutes the CO₂ produced from labeled acetate. This may be explained by compartmentation or preferred pathways for the disposition of acetyl CoA derived from different sources.     

AMPK and metabolic adaptation by the heart to pressure overload

Accelerated glycolysis in hypertrophied hearts may be a compensatory response to reduced energy production from long-chain fatty acid oxidation with 5'-AMP-activated protein kinase (AMPK) functioning as a cellular signal. Therefore, we tested the hypothesis that enhanced fatty acid oxidation improves energy status and normalizes AMPK activity and glycolysis in hypertrophied hearts. Glycolysis, fatty acid oxidation, AMPK activity, and energy status were measured in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. Hearts from halothane (3-4%)-anesthetized rats were perfused with KH solution containing either palmitate, a long-chain fatty acid, or palmitate plus octanoate, a medium-chain fatty acid whose oxidation is not impaired in hypertrophied hearts. Compared with control, fatty acid oxidation was lower in hypertrophied hearts perfused with palmitate, whereas it increased to similar values in both groups with octanoate plus palmitate. Glycolysis was accelerated in palmitate-perfused hypertrophied hearts and was normalized in hypertrophied hearts by the addition of octanoate. AMPK activity was increased three- to sixfold with palmitate alone and was reduced to control values by octanoate plus palmitate. Myocardial energy status improved with the addition of octanoate but did not differ between groups. Our findings, particularly the correspondence between glycolysis and AMPK activity, provide support for the view that activation of AMPK is responsible, in part, for the acceleration of glycolysis in cardiac hypertrophy. Additionally, they indicate myocardial AMPK is activated by energy state-independent mechanisms in response to pressure overload, demonstrating AMPK is more than a sensor of the heart's energy status.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.     

Oxidative metabolism of long-chain fatty acids in mitochondria from sheep and rat liver. Evidence that sheep conserve linoleate by limiting its oxidation.

Mitochondria isolated from the livers of sheep and rats were shown to oxidize palmitate, oleate and linoleate in a tightly coupled manner, by monitoring the oxygen consumption associated with the degradation of these acids in the presence of 2mM-L-malate. Rat liver mitochondria oxidized linoleate and oleate at a rate 1.2-1.8 times that of palmitate. Sheep liver mitochondria had a specific activity for the oxidation of palmitate that was 50-80% of that of rats and a specific activity for the oxidation of oleate and linoleate that was 30-40% that of rats. This would indicate that sheep conserved linoleate by limiting its oxidation. Carnitine acyltransferase I (CAT I) actively esterified palmitoyl-CoA and linoleate to carnitine in both rat and sheep liver mitochondria, and in both cases the rate for linoleate was faster than for palmitate. The CAT I reaction in both rat and sheep liver was inhibited by micromolar amounts of malonyl-CoA. With 90 microM-palmitoyl-CoA as substrate, CAT I was inhibited by 50% with 2.5 microM-malonyl-CoA in rats, and in sheep, 50% inhibition was found with all malonyl-CoA concentrations tested (1-5 microM). With 90 microM-linoleate as substrate for CAT I, a much larger difference in response to malonyl-CoA was seen, the rat enzyme being 50% inhibited at 22 microM-malonyl-CoA, whereas sheep liver CAT I was 91% and 98% inhibited at 1 microM- and 5 microM-malonyl-CoA respectively. We propose that malonyl-CoA may act as an important regulator of beta-oxidation in sheep, discriminating against the use of linoleate as an energy-yielding substrate.     

Effects of valproic acid on cardiac metabolism

We investigated whether the antiepileptic valproic acid (VPA) might interfere with oxidative metabolism in heart, as it does in liver. We administered VPA to working rat hearts perfused with radiolabeled carbohydrate and fatty acid fuels. Measurements included oxidation rates of (i) glucose, pyruvate, or lactate in the presence of palmitate and (ii) palmitate, octanoate, or butyrate in the presence of glucose. Oxidation rates were quantified as the rate of appearance of 14CO2 or 3H2O from 14C- or 3H-labeled substrates. In hearts perfused with palmitate, VPA (1 mmol/L) strongly inhibited the oxidation of pyruvate and lactate but slightly stimulated the oxidation of glucose. VPA also inhibited lactate or pyruvate uptake into erythrocytes in vitro. In hearts perfused with glucose, VPA strongly inhibited the oxidation of palmitate and octanoate but had no effect on butyrate oxidation. The absence of valproate CoA ligase activity in cell-free homogenates indicated that the inhibition of fatty acid oxidation by VPA did not require prior activation to valproyl-CoA. The results are consistent with the hypothesis that VPA selectively interferes with myocardial fuel oxidation by mechanisms that are independent of conversion to the CoA thioester.     

Effects of hypoxia and reoxygenation on adult rat heart cell metabolism

Isolated adult rat heart cells were used to study the effects of oxygen deprivation followed by reoxygenation upon myocardial metabolism. Calcium-tolerant nonbeating myocytes were incubated for 5, 30, or 60 min under 100% oxygen or 100% nitrogen and then rinsed with oxygenated buffer. Substrate oxidation was studied by incubating the cells with 14C-labeled glucose, pyruvate, or octanoate and determining the rates of 14CO2 production from the individual substrates. After 5 min of hypoxia, metabolism of glucose, as assessed by glucose oxidation and lactate production, was significantly depressed. Pyruvate and octanoate oxidation were unaltered. Oxygen consumption was also unchanged by short-term hypoxia and reoxygenation. With reoxygenation after 30 min of oxygen deprivation, more exaggerated changes in glucose metabolism were noted as well as a depression in pyruvate oxidation and unaltered octanoate oxidation. Oxidation of octanoate was slightly depressed after 60 min of hypoxia. Cell viability assessed after reoxygenation was not significantly altered until 60 min of oxygen deprivation. The results indicate that cytosolic changes occur after short periods of hypoxia followed by reoxygenation, whereas mitochondrial function is more resistant to damage inflicted by hypoxia and reoxygenation.     

Effects of oxygen radicals on substrate oxidation by cardiac myocytes

Freshly isolated adult rat heart cells were used to study the effects of oxygen-free radicals on the myocardial oxidation of different substrates. The calcium-tolerant quiescent cells were incubated with xanthine plus xanthine oxidase as the source of free radicals. The oxidation of exogenous glucose, lactate and octanoate was severely inhibited (approx. 70%) by products of xanthine oxidase activity. Superoxide dismutase plus catalase effectively prevented the inhibition of oxidation. Cellular high energy phosphate levels were decreased in the presence of the oxygen free radical generating system although cell viability determined by Trypan blue exclusion and light microscopic assessment of normal morphology was not affected. These data suggest that oxygen free radicals decrease myocardial substrate oxidation which may contribute to the functional and ultrastructural changes in the myocardium under conditions such as reoxygenation after hypoxia and reperfusion after ischemia.     

Sodium benzoate inhibits fatty acid oxidation in rat liver: effect on ammonia levels

Sodium benzoate inhibited PC and octanoic acid-mediated State 3 respiration rates by 39 and 29%, respectively, at 0.5 mM in isolated rat liver mitochondria. At 2 mM, benzoate did not affect State 3 respiration rates with either succinate or malate plus glutamate, indicating that it did not act as an uncoupler. The oxidation of palmitate and octanoate was inhibited by 39 and 54% at 2 mM benzoate in liver homogenates. Benzoate, at 10 mmol/kg caused significant decreases in the levels of hepatic ATP, CoA, and acetyl-CoA. Administration of sodium benzoate to rats caused a dose-dependent increase in hepatic ammonia levels. However, the inhibitory effect of benzoate on fatty acid oxidation is not mediated through ammonia since ammonium chloride, at 1 mM, did not inhibit PC or octanoate oxidation in mitochondria or their oxidation in liver homogenate. Our results warrant a reevaluation of the use of sodium benzoate in the treatment of hyperammonemia.     

Regulation of carbohydrate and fatty acid utilization by L-carnitine during cardiac development and hypoxia

This study is designed to investigate whether substrate preference in the myocardium during the neonatal period and hypoxia-induced stress is controlled intracellularly or by extracellular substrate availability. To determine this, the effect of exogenous L-carnitine on the regulation of carbohydrate and fatty acid metabolism was determined during cardiac stress (hypoxia) and during the postnatal period. The effect of L-carnitine on long chain (palmitate) and medium chain (octonoate) fatty acid oxidation was studied in cardiac myocytes isolated from less than 24 h old (new born; NB), 2 week old (2 week) and hypoxic 4 week old (HY) piglets. Palmitate oxidation was severely decreased in NB cells compared to those from 2 week animals (0.456 ± 0.04 vs. 1.207 ± 0.52 nmol/mg protein/30 min); surprisingly, cells from even older hypoxic animals appeared shifted toward the new born state (0.695 ± 0.038 nmol/mg protein/30 min). Addition of L-carnitine to the incubation medium, which stimulates carnitine palmitoyl-transferase I (CPTI) accelerated palmitate oxidation 3 fold in NB and approximately 2 fold in HY and 2 week cells. In contrast, octanoate oxidation which was greater in new born myocytes than in 2 week cells, was decreased by L-carnitine suggesting a compensatory response. Furthermore, oxidation of carbohydrates (glucose, pyruvate, and lactate) was greatly increased in new born myocytes compared to 2 week and HY cells and was accompanied by a parallel increase in pyruvate dehydrogenase (PDH) activity. The concentration of malonyl-CoA, a potent inhibitor of CPTI was significantly higher in new born heart than at 2 weeks. These metabolic data taken together suggest that intracellular metabolic signals interact to shift from carbohydrate to fatty acid utilization during development of the myocardium. The decreased oxidation of palmitate in NB hearts probably reflects decreased intracellular L-carnitine and increased malonyl-CoA concentrations. Interestingly, these data further suggest that the cells remain compliant so that under stressful conditions, such as hypoxia, they can revert toward the neonatal state of increased glucose utilization.     

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.     

The control of fatty acid metabolism in liver cells from fed and starved sheep.

Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.     

Regular exercise is associated with a protective metabolic phenotype in the rat heart

Adaptation of myocardial energy substrate utilization may contribute to the cardioprotective effects of regular exercise, a possibility supported by evidence showing that pharmacological metabolic modulation is beneficial to ischemic hearts during reperfusion. Thus we tested the hypothesis that the beneficial effect of regular physical exercise on recovery from ischemia-reperfusion is associated with a protective metabolic phenotype. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured in isolated working hearts from sedentary control (C) and treadmill-trained (T: 10 wk, 4 days/wk) female Sprague-Dawley rats submitted to 20 min ischemia and 40 min reperfusion. Training resulted in myocardial hypertrophy (1.65 +/- 0.05 vs. 1.30 +/- 0.03 g heart wet wt, P < 0.001) and improved recovery of function after ischemia by nearly 50% (P < 0.05). Glycolysis was 25-30% lower in T hearts before and after ischemia (P < 0.05), whereas rates of glucose oxidation were 45% higher before ischemia (P < 0.01). As a result, the fraction of glucose oxidized before and after ischemia was, respectively, twofold and 25% greater in T hearts (P < 0.05). Palmitate oxidation was 50-65% greater in T than in C before and after ischemia (P < 0.05), whereas lactate oxidation did not differ between groups. Alteration in content of selected enzymes and proteins, as assessed by immunoblot analysis, could not account for the reduction in glycolysis or increase in glucose and palmitate oxidation observed. Combined with the studies on the beneficial effect of pharmacological modulation of energy metabolism, the present results provide support for a role of metabolic adaptations in protecting the trained heart against ischemia-reperfusion injury.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

Regulation of intermediary metabolism in rat cardiac myocyte by extracellular glycerol

In the human heart, although all substrates compete for energy production, fatty acids (FA) represent the main substrate for ATP production. In the healthy heart, a balance between FA and carbohydrate utilization ensures that energy supply matches demand. This study was carried out to evaluate, in a model of spontaneously beating neonatal rat cardiomyocytes in culture, the hypothesis that glycerol could play a central role in the metabolic control of the routes involving long chain FAs and may then affect the balance between beta-oxidation and glucose oxidation. The intracellular-free glycerol significantly increased with extracellular glycerol concentration (0 to 660 microM). The synthesis of phospholipids was significantly increased in parallel with both extracellular glycerol (1.5 and 14.8 nmol glycerol/mg protein, at 82 and 660 microM of extracellular glycerol, respectively). The oxidation of glycerol increased proportionally to extracellular glycerol concentration (from 1 to 3 nmol glycerol/mg protein, at 82 microM and 660 microM extracellular glycerol, respectively, P<0.001). At its maximum, this oxidation represented 15% of the glucose oxidation, which was not affected by glycerol extracellular supply or intracellular availability. Conversely, extracellular glycerol significantly reduced the palmitate oxidation above (-47% at 660 microM glycerol), but not octanoate oxidation. Investigations on the mechanism of the decreased palmitate oxidation reveals a glycerol-dependent increase in malonyl-CoA associated with a significant decrease in CPT-1 activity which accounts for the difference between palmitate and octanoate. These results clearly demonstrate the importance of glycerol in regulating the cardiac metabolic pathways and energy balance.     

Stimulation of fatty acid oxidation in myocytes by phosphodiesterase inhibitors and adenosine analogues.

The effect of various phosphodiesterase inhibitors, and adenosine analogues on palmitate oxidation, were studied in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a concentration of 250 microM, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. The specific A1-agonist, N6-cyclopentyladenosine, increased palmitate oxidation by 56%, at a concentration of 250 microM. Moreover, the nucleoside transport inhibitor, S-(P-Nitrobenzyl-)6-thioinosine, increased palmitate oxidation by 40%, at a concentration of 100 microM. These data suggest that the stimulation of palmitate oxidation by enoximone and adenosine analogues may be mediated via the inhibition of the uptake and/or the oxidation of glucose in myocytes.     

An accurate and sensitive assay of [14C]octanoate oxidation and its application on tissue homogenates and fibroblasts

A procedure was developed to assay [14C]octanoate oxidation from the production of both 14CO2 and 14C-labeled acid-soluble products. Octanoic acid and its CoA and carnitine esters were eliminated from the acid-soluble products by alkaline hydrolysis of the esters and acidification and binding of the acid to Lipidex 1000. The method was evaluated with homogenates of various rat tissues and human muscles and with human fibroblasts. 14CO2 production was variable and comprised less than 3% of the total oxidation products with homogenates and 26 +/- 19% with fibroblasts. As compared to palmitate, oxidation rates of octanoate were higher in rat liver and heart homogenates, of the same magnitude in muscle homogenates, but lower in fibroblasts. The proportion of antimycin-insensitive oxidation was much lower with octanoate than with palmitate. Using the assay a case of medium-chain acyl-CoA dehydrogenase deficiency could be indicated.     

Fatty acid and glucose oxidation by cultured rat heart cells

Monolayer cultures of fetal rat myocardial cells can be utilized to examine substrate preferences and interactions. The specific activity of glucose oxidation by myocardial cell cultures was high in sparse cultures but decreased with increased cell density. In contrast, palmitate oxidation was independent of initial cell density. Palmitate inhibited glucose oxidation by 50% in rat heart cultures. Glucose had only a slight sparing effect on palmitate oxidation. This suggests that fetal and newborn rat myocardial cells in culture preferentially oxidize palmitate similar to adult heart. The sparing effect of palmitate on glucose oxidation is accounted for by inhibition of the glycolytic-aerobic pathway and not by inhibition of the pentose phosphate pathway. Data on oxidation of ¹⁴C-pyruvate specifically labelled suggest that palmitate or a product of its oxidation such as acetyl-CoA may be acting directly to inhibit the pyruvate dehydrogenase complex. Palmitate oxidation per mg of cell protein was constant from 15 days gestational age to 2 days postnatal age. The observed differences between cultured cells and the intact heart may relate to decreased aerobic metabolism in monolayer cell culture and suggest that the increase in fatty acid oxidation observed in vivo is controlled by the oxygen environment of the cell. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information similar to an adult organ perfusion model.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.