Influence of pH of the medium on free fatty acid utilization by isolated mammalian cells

Studies with Ehrlich ascites tumor cells showed that small decreases in the pH of the incubation medium from 7.4 increase the magnitude of incorporation of free fatty acid (FFA) into the cells from an albumin solution. A similar effect occurred when rabbit erythrocytes, rat heart slices, or rat liver slices were incubated with FFA-bovine albumin solutions and when tumor cells were incubated with FFA in media containing human albumin, -lactoglobulin, or rat plasma. The effect was not seen when the medium contained no protein. When the pH of the albumin-containing medium was lowered from 7.4 to 6.6, oxidation of FFA to CO(2) by the tumor cells increased, esterification of the FFA (mostly into phospholipids and triglycerides) increased, and less esterified radio-active fatty acid was depleted from the cells. Hence, more fatty acid accumulated in the cells in more acid media. These findings suggest that small changes in extracellular pH might regulate FFA utilization and lipid accumulation in mammalian tissues.     

Effect of fatty acid-binding proteins on intermembrane fatty acid transport studies on different types and mutant proteins.

Liposomes of different charge fixed to nitrocellulose filters were used to study the transfer of fatty acids to rat heart or liver mitochondria in the presence of fatty acid-binding protein (FABP) or albumin. [14C]Palmitate oxidation was used as a parameter. Different FABP types and heart FABP mutants were tested. The charge of the liposomes did not influence the solubilization and mitochondrial oxidation of palmitate without FABP and the amount of solubilized palmitate in the presence of FABP. Mitochondria did not show a preference for oxidation of FABP-bound palmitate over their tissue-specific FABP type. All FABP types increased palmitate oxidation by heart and liver mitochondria with neutral, positive and negative liposomes by 2.5-fold, 3.2-fold and twofold, respectively. Ileal lipid-binding protein and H-FABP mutants that do not bind fatty acid had no effect. Other H-FABP mutants had different effects, dependent on the site of mutation. The effect of albumin was similar to, but not dependent on, liposome charge. The ionic strength had only a slight effect. In conclusion, the transfer of palmitate from liposomal membranes to mitochondria was increased by all FABP types to a similar extent. The membrane charge had a large effect in contrast to the origin of the mitochondria.     

Regulation of long chain fatty acid activation in heart muscle.

Regulation of fatty acid activation was studied in whole tissue homogenates of rat heart. The palmityl-CoA synthestase activity was proportional to the fatty acid to albumin ratio in the incubation medium with maximal activity occurring at a molar ratio of about 5. Fatty acyl-CoA synthetase activity was inhibited by products of the reaction (AMP, pyrophosphate, and palmityl-CoA). The apparent Ki for palmityl-CoA inhibition was 5 muM and this inhibition could be relieved by CoA-SH or albumin. The Km for CoA-SH in the absence of palmityl-CoA was 7 muM and was increased to 24 muM by addition of 8 muM palmityl-CoA. Cytosolic and mitochondrial levels of CoA-SH and carnitine were estimated in whole tissue homogenates of heart and liver. From 90 to 100% of whole tissue CoA was recovered in the mitochondrial fraction of heart muscle and it was estimated that the cytosolic concentration of free CoA-SH probably never exceeds its Km value for fatty acid activation in this tissue. Therefore, the rate of fatty acid activation would be expected to depend on the availability of CoA-SH in the cytosolic space. By adjusting the concentration of CoA-SH in the cytosol to the rate of acetyl-CoA oxidation, carnitineacetyl-CoA transferase may function in cardiac muscle to couple the rate of fatty acid activation in the cytosolic compartment to acetyl-CoA oxidation in the mitochondria. Approximately 30% of whole tissue CoA-SH was located in the cytosolic space in liver. Heart muscle has about twice as much carnitine as liver but in both tissues 100% of whole tissue carintine was located in the cytosolic space. The ratio of carnitine to CoA-SH in the cytosolic space was estimated to be about 100 in heart and 17 in liver. This high ratio in cardiac muscle may function to channel fatty acids toward oxidation rather than toward synthesis of complex lipids.     

Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.

Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.     

Immunochemical analysis of the peroxisomal beta-oxidation enzymes in rat and human heart and skeletal muscle and in skeletal muscle of Zellweger patients

Immunoblot analyses with antibodies against the peroxisomal beta-oxidation enzymes from rat liver showed the presence of these enzymes in rat and human liver and kidney and rat adrenal gland. The bifunctional protein could not be detected in muscle tissues or cultured muscle cells. Acyl-CoA oxidase was detected in rat heart and cultured human muscle cells. 3-Ketoacyl-CoA thiolase was also detected in human and rat heart and skeletal muscle; however, this enzyme was not detectable in skeletal muscle of Zellweger patients, in agreement with the absence of peroxisomal fatty acid oxidation.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Palmitic acid uptake by the rat soleus muscle in vitro.

Abstract: The rate of fatty acid uptake, oxidation, and deposition in skeletal muscles in relation to total and unbound to albumin fatty acids concentration in the medium were investigated in the incubated rat soleus muscle. An immunohistochemical technique was applied to demonstrate whether the albumin-bound fatty acid complex from the medium penetrates well within all areas of the muscle strips. It was found that the percentage of incorporation of palmitic acid into intramuscular lipids was fairly constant, independently of the fatty acid concentration in the medium, and amounted to 63-72% for triacylglycerols, 7-12% for diacylglycerols-monoacylglycerols, and 19-26% for phospholipids. Both palmitic acid incorporation into the muscle triacylglycerol stores and its oxidation to CO2 closely correlated with an increase in both total and unbound to albumin fatty acid concentrations in the incubation medium. Under conditions of increased total but constant unbound to albumin palmitic acid concentrations, the incorporation of palmitic acid into triacylglycerols and its oxidation to CO2 were also increased, but to a lower extent. This supports the hypothesis that the cellular fatty acid metabolism depends not only on the availability of fatty acids unbound to albumin, but also on the availability of fatty acids complexed to albumin.     

Metabolism of triglyceride fatty acid by the perfused rat heart

1. Chyle lipids, labelled with (14)C, are taken up and oxidized by the isolated perfused rat heart. 2. In recirculatory perfusions, when chyle lipids are the sole exogenous energy source, about 24% of the total oxygen uptake is accounted for by their oxidation. This proportion is not changed by starvation of the rats for 48hr. and falls when an external work load is imposed on the left ventricle. 3. With albumin in the perfusion medium, the rate of (14)CO(2) output is reduced by half and there is a rise in the proportion of (14)C-labelled free fatty acids in the medium. 4. Clearing-factor lipase appears in the perfusion medium when chyle lipids are perfused through the heart. In the absence of albumin, the activity of the medium enzyme is low and only a small proportion of the (14)CO(2) output can be accounted for by the oxidation of free fatty acids released by it. In the presence of albumin, the enzyme is more active in the medium. 5. When a substantial proportion of the total clearing-factor lipase is removed from the heart by a prior perfusion with heparin, (14)C-labelled chyle lipid perfused subsequently is oxidized at only half the normal rate.     

Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.     

Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.     

Oxidation of oleic and elaidic acids in rat and human heart homogenates

Parallel incubations with uniformly 14C-labeled oleic and elaidic acids were conducted to compare oxidation rates in tissue homogenates prepared from rat and human hearts. Radioactivity in 14CO2 and 14C-labeled chain-shortened acid-soluble products was used to measure the extent of oxidation. Oxidation rates (pmol/min per mg heart protein) determined on 14C-labeled acid-soluble products suggest that oleic acid was oxidized 35-40% faster than elaidic acid by both male and female rat heart homogenates, whereas human heart homogenates oxidized these fatty acids at equal rates. Rates for female heart homogenates were somewhat higher than those for males in rats and humans. Rates of formation of 14CO2 were the same for each acid in rat and human heart tissue. Comparative rates of formation of oxidation products expressed as oleic/elaidic ratios from parallel incubations confirm that preferential oxidation of oleic acid occurred with rat heart homogenates, but not with the human heart homogenates. These data suggest that the presence of the trans double bond in elaidic acid does not impair its utilization for energy by human heart muscle.     

Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We (34) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

Undecanesulfonate does not allosterically activate H+ uniport mediated by uncoupling protein-1 in brown adipose tissue mitochondria

Undecanesulfonate is transported by uncoupling protein-1. Its inability to induce H+ uniport with reconstituted uncoupling protein-1 supports fatty acid cycling hypothesis. Rial et al. [Rial, E., Aguirregoitia, E., Jimenez-Jimenez, J., & Ledesma, A. (2004). Alkylsulfonates activate the uncoupling protein UCP1: Implications for the transport mechanism. Biochimica et Biophysica Acta, 1608, 122-130], have challenged the fatty acid cycling by observing uncoupling of brown adipose tissue mitochondria due to undecanesulfonate, interpreted as allosteric activation of uncoupling protein-1. We have estimated undecanesulfonate effects after elimination of endogenous fatty acids by carnitine cycle in the presence or absence of bovine serum albumin. We show that the undecanesulfonate effect is partly due to fatty acid release from albumin when undecanesulfonate releases bound fatty acid and partly represents a non-specific uncoupling protein-independent acceleration of respiration, since it proceeds also in rat heart mitochondria lacking uncoupling protein-1 and membrane potential is not decreased upon addition of undecanesulfonate without albumin. When the net fatty acid-induced uncoupling was assayed, the addition of undecanesulfonate even slightly inhibited the uncoupled respiration. We conclude that undecanesulfonate does not allosterically activate uncoupling protein-1 and that fatty acid cycling cannot be excluded on a basis of its non-specific effects.     

Palmityl-CoA oxidase: Detection in several guinea pig tissues and peroxisomal localisation in mucosa of small intestine

Using a sensitive assay palmityl-CoA oxidase activity was detected in several guinea pig tissues including liver, kidney, small intestine, lung, muscle, spleen and heart. Subcellular fractionation using sucrose and metrizamide gradients demonstrated that in small intestinal mucosa palmityl-CoA oxidase is localised in the same particles as catalase. These observations are discussed in the context of a role for peroxisomal fatty acid oxidation in energy balance and thermogenesis.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid on pyruvate dehydrogenase complex activity in starved and alloxan-diabetic rats

Intravenous administration of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid had no effect on the proportion of pyruvate dehydrogenase complex in the active form in heart, diaphragm or gastrocnemius muscles or in liver, kidney or adipose tissue of fed normal rats. The compound reversed the effect of 48h starvation (which decreased the proportion of active complex) in heart muscle, partially reversed the effect of starvation in kidney, but had no effect in the other tissues listed. The compound failed to reverse the effect of alloxan-diabetes (which decreased the proportion of active complex) in any of these tissues. In perfused hearts of fed normal rats, 2-tetradecylglycidate reversed effects of palmitate (which decreased the proportion of active complex), but it had no effect in the absence of palmitate. In perfused hearts of 48h-starved rats the compound increased the proportion of active complex to that found in fed normal rats in the presence or absence of insulin. In perfused hearts of diabetic rats the compound normalized the proportion of active complex in the presence of insulin, but not in its absence. Palmitate reversed the effects of 2-tetradecylglycidate in perfused hearts of starved or diabetic rats. Evidence is given that 2-tetradecylglycidate only reverses effects of starvation and alloxan-diabetes on the proportion of active complex in heart muscle under conditions in which it inhibits fatty acid oxidation. It is concluded that effects of starvation and alloxan-diabetes on the proportion of active complex in heart muscle are dependent on fatty acid oxidation. Insulin had no effect on the proportion of active complex in hearts or diaphragms of fed or starved rats in vitro. In perfused hearts of alloxan-diabetic rats, insulin induced a modest increase in the proportion of active complex in the presence of albumin, but not in its absence.     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.     

Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis

The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.     

Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs

Hearts from 4 week-old weanling pigs were capable of continuous work output when perfused with Krebs-Henseleit buffer containing 11 mM glucose. Perfused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of glucose plus physiological concentrations (0.1 mM) of either palmitate or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and carnitine. When hearts were perfused with 1 mM fatty acids, the work output declined below control values. Erucic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by perfused rat hearts. In addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated perfused pig hearts used in the present study, erucic acid oxidation rates were as high as palmitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during erucic acid perfusion did not change noticeably from those during glucose perfusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to erucic acid are not replicated in the intact pig heart.