beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

Effects of lactation of ketogenesis from oleate or butyrate in rat hepatocytes.

1. Rates of ketogenesis from endogenous butyrate or oleate were measured in isolated hepatocytes prepared from fed rats during different reproductive states [virgin, pregnant, early-lactating (2-4 days) and peak-lactating (10-17 days)]. In the peak-lactation group there was a decrease (25%) in the rate of ketogenesis from butyrate, but there were no differences in the rates between the other groups. Wth oleate, the rate of ketogenesis was increased in the pregnant and in the early-lactation groups compared with the virgin group, whereas the rate was 50% lower in the peak-lactation group. 2. Experiments with [1-(14)C]oleate indicated that these differences in rates of ketogenesis were not due to alterations in the rate of oleate utilization, but to changes in the amount of oleoyl-CoA converted into ketone bodies. 3. Although the addition of carnitine increased the rates of ketogenesis from oleate in all groups of rats, it did not abolish the differences between the groups. 4. Measurements of the accumulation of glucose and lactate showed that hepatocytes from rats at peak lactation had a higher rate of glycolytic flux than did hepatocytes from the other groups. After starvation, the rate of ketogenesis from oleate was still lower in the peak-lactation group compared with the control group. This suggests that the alteration in ketogenic capacity in the former group is not merely due to a higher glycolytic flux. 5. It is concluded that livers from rats at peak lactation have a lower capacity to produce ketone bodies from long-chain fatty acids which is due to an alteration in the partitioning of long-chain acyl-CoA esters between the pathways of triacylglycerol synthesis and beta-oxidation. The physiological relevance of this finding is discussed.     

Lipid metabolism in microsomal fraction from photosynthetic tissue. Effects of catalase and hydrogen peroxide on oleate desaturation.

On incubation of microsomal fraction from pea (Pisum sativum L.) leaves with ammonium [1-14C]oleate or [1-14C]oleoyl-CoA in the presence of ATP, CoA, Mg2+ and NADH, the major reactions observed were those catalysed by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The reaction catalysed by oleoyl phosphatidylcholine desaturase was specifically inhibited by H2O2, and this inhibitory effect was overcome by catalase (EC 1.11.1.6).     

Influence of fasting on the effects of ischemic preconditioning in the ischemic-reperfused rat heart

The effects of fasting and ischemic preconditioning (IP) on heart function of Langendorff-perfused rat hearts exposed to 25 min global ischemia plus 30 min reperfusion (RP), were correlated with lactate release and tissue-levels of long-chain acyl carnitine (LCCa) and CoA (LCCoA). IP was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. Creatine kinase leakage was measured to assess the extent of cardiac injury. Fasting reduced the ischemic-induced contracture, improved RP recovery of mechanical function, reduced lactate release and increased the end-ischemia LCCoA and LCCa levels. Both in the fed and the fasted rat hearts IP delayed the pacemaker depression, reduced the amplitude of ischemic contracture and improved the RP recovery of contraction. However, IP reduced creatine kinase and lactate release only in the fed rat hearts. IP had no effects on tissue LCCa and LCCoA in both groups. These data suggest that: 1) beneficial effects of fasting may be ascribed, at least in part, to a reduced lactate production which may attenuate ischemic myocyte acidification and to the accumulation of fatty acyl esters which would favour citric acid cycle replenishment during RP. 2) beneficial effects of IP could be in part explained by the reduction of lactate production in the fed group although data obtained with the fasted rat heart indicate that another mechanisms must also be involved in the effects of IP. 3) accumulation of LCCoA and LCCa is not involved in the noxious effects of ischemia as well as in the protection effected by IP.     

Intermediates of myocardial mitochondrial beta-oxidation: possible channelling of NADH and of CoA esters

Adult rat heart mitochondria were isolated and incubated with [U-14C]hexadecanoyl-CoA or unlabelled hexadecanoyl-CoA. The accumulating CoA and carnitine esters and [NAD+]/[NADH] ratio were measured by HPLC or tandem mass spectrometry. Despite minimal changes in the intramitochondrial [NAD+]/[NADH] ratio, 2, 3-unsaturated and 3-hydroxyacyl esters were observed as well as saturated acyl-CoA and acylcarnitine esters. In addition to acetylcarnitine, significant amounts of butyryl-, hexanoyl-, octanoyl- and decanoylcarnitines were detected and measured. Rat myocardial beta-oxidation is subject to control at the level of 3-hydroxyacyl-CoA dehydrogenase but this control is not due to a simple lack of oxidised NAD. We hypothesise a pool of NAD in contact between the trifunctional protein of beta-oxidation and complex I of the respiratory chain, the turnover of which is responsible for some of the control of beta-oxidation flux. In addition, short- and medium-chain acylcarnitine esters were detected whereas only small amounts of long-chain acylcarnitines were present. This may imply the presence of a mitochondrial carnitine octanoyl transferase or may reflect channelling of long-chain CoA esters so that they are not available for carnitine palmitoyl transferase II activity.     

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.     

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.     

Effect of starvation and diabetes on the sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate.

The sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate was decreased markedly in liver mitochondria isolated from either 48 h-starved or streptozotocin-diabetic rats. These treatments of the rat also decreased the sensitivity of fatty acid oxidation by isolated hepatocytes to inhibition by this compound. Furthermore, incubation of hepatocytes prepared from fed rats with N6O2'-dibutyryl cyclic AMP also decreased the sensitivity, whereas incubation of hepatocytes prepared from starved rats with lactate plus pyruvate had the opposite effect on 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation. The sensitivity of carnitine palmitoyltransferase I of mitochondria to 4-hydroxyphenylglyoxylate increased in a time-dependent manner, as previously reported for malonyl-CoA. Likewise, oleoyl-CoA activated carnitine palmitoyltransferase I in a time-dependent manner and prevented the sensitization by 4-hydroxyphenylglyoxylate. Increased exogenous carnitine caused a moderate increase in fatty acid oxidation by hepatocytes under some conditions and a decreased 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation at low oleate concentration, without decreasing the difference in 4-hydroxyphenylglyoxylate inhibition between fed- and starved-rat hepatocytes. Time-dependent changes in the conformation of carnitine palmitoyltransferase I or the membrane environment may be involved in differences among nutritional states in 4-hydroxyphenylglyoxylate-sensitivity of carnitine palmitoyltransferase I.     

Carnitine metabolism in livers and hearts of 48h-starved rats: the contribution of fat and carbohydrate to acylcarnitine formation

The work investigated the effects of administration of 2-tetradecylglycidate (TDG), an inhibitor of mitochondrial long-chain fatty acid oxidation, alone or in combination with glucose, on concentrations of free and acylated carnitine in livers and hearts of 48 h-starved rats. The only significant effect of TDG in the heart was to decrease [short-chain acylcarnitine]. This demonstrates that in heart, fat oxidation is linked to the formation of short-chain acylcarnitine. Cardiac [short-chain acylcarnitine] was not significantly decreased by TDG if the rats were also administered glucose, suggesting that acyl CoA derived from glucose may be used for short-chain acylcarnitine formation in TDG-treated rats. TDG significantly decreased in [free carnitine]. No changes in [short-chain acylcarnitine] were observed. This indicates that formation of short-chain acylcarnitine in liver is not determined by the rates of fat oxidation. It was calculated that at least 63% of the acyl-groups esterified to carnitine were generated by intramitochondrial beta-oxidation. The effects of glucose and TDG on hepatic concentrations of free and long-chain acylcarnitine were additive, suggesting that extramitochondrial fat oxidation can contribute to acylcarnitine formation in liver.     

A single therapeutic dose of valproate affects liver carbohydrate, fat, adenylate, amino acid, coenzyme A, and carnitine metabolism in infant mice: possible clinical significance

We have previously reported that chronic valproate administration reduced ketonemia in suckling mice and fasting epileptic children. The present study demonstrates that even a single dose of valproate in the therapeutic range for man caused a prolonged reduction of plasma beta-hydroxybutyrate levels in normal infant mice; the plasma glucose concentration was also significantly lowered. In the livers of these animals, there were extraordinary decreases in levels of free coenzyme A, acetyl CoA and free carnitine. Concomitantly concentrations of acid-soluble fatty acid (short-chain, non-acetyl) coenzyme A esters and of acid-insoluble (long-chain) fatty acid carnitine esters increased. There was evidence for inhibition of the metabolic flux through the Krebs citric acid cycle at those enzyme reactions which require coenzyme A. While valproate doubled liver alanine levels, concentrations of liver aspartate, glutamate and glutamine were reduced. All of the valproate-induced metabolite changes can be explained by the decrease of coenzyme A due to the accumulation of acid-soluble (non-acetyl) coenzyme A esters (presumably valproyl CoA and further metabolites). Decreased coenzyme A would limit the activities of one or more enzymes in the pathway of fatty acid oxidation and the Krebs citric acid cycle. Secondary decreases in acetyl CoA would limit both ketogenesis and gluconeogenesis. Decreased levels of selected hepatic amino acids could reflect their use as alternative fuels. The effect of clinical doses of valproate in infant mice may relate to the valproate-associated syndrome of hepatic failure and Reye-like encephalopathy in some infants and children and suggest a simple screen for those who may be at particular risk.     

Metabolism of pyruvate and carnitine esters in bovine epididymal sperm mitochondria

Filipin-treated bovine epididymal spermatozoa have been used to study mitochondrial l-acetylcarnitine, l-palmitoylcarnitine, and pyruvate metabolism. The cells were supplemented with malate to allow rapid rates of substrate oxidation. The rate of l-palmitoylcarnitine-supported state 3 respiration was slow. In contrast, pyruvate, acetylcarnitine, or lactate supported rapid and approximately equal respiratory rates. l-Palmitoylcarnitine was a weak inhibitor of pyruvate-supported respiration and pyruvate use and a more potent inhibitor of l-acetylcarnitine. l-Carnitine was an effective inhibitor of l-acetylcarnitine oxidation; however, it did not influence l-palmitoylcarnitine oxidation or inhibit pyruvate utilization. Pyruvate (1.4 mm) disappearance was rapid and was complete within 6–7 min; the lactate produced during pyruvate metabolism was then oxidized. ATP synthesis was constant throughout the 20-min incubation. With pyruvate plus l-acetylcarnitine as substrate, the l-acetylcarnitine concentration initially dropped and then recovered to a level that was dependent on free carnitine addition. Data obtained from experiments using [2-¹⁴C]pyruvate indicated that the ¹⁴C label from pyruvate and lactate entered the l-acetylcarnitine pool and labeling was maximal when free l-carnitine was added. The rate of citrate synthesis was maximal when pyruvate was being metabolized; the largest total accumulation occurred when all three substrates were included in the incubation. The data suggest that the high NAD⁺/ NADH maintained during pyruvate metabolism may restrict flux through the citric acid cycle. The relationships of l-carnitine and the l-carnitine esters to pyruvate metabolism are discussed.     

Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine acetyltransferase and [total carnitine] in liver are closely related.     

Carnitine worsens both injury and recovery of contractile function after transient ischemia in perfused rat heart

While carnitine overload appears to have therapeutic effects in pathological situations such as heart recovery after ischemia, its benefits as dietary supplementation for aerobic exercise have been questioned. We studied the effect of carnitine supplementation on the response of perfused rat heart to ischemia and reperfusion. Supplementation of the perfusion medium with 1 mM carnitine had no effect on cardiac performance in normoxic hearts, although it lowered lactate production by nearly 80%. Carnitine did not affect the amount of lactate accumulated during 30 min of ischemia, which was recovered in the perfusate immediately after reperfusion. However, carnitine worsened tissue injury, as shown by the 70% increase in creatine kinase release. Carnitine also worsened the recovery of contractile function, as revealed by the slower increase in heart rate and contractile force. In addition, carnitine supplementation increased contracture of the heart shortly after reperfusion. Therefore, in conditions where it does not increase glucose oxidation, carnitine supplementation worsens both injury and recovery of contractile function after transient ischemia in perfused rat heart.     

Effects of fasting and hypoxic preconditioning on the hypoxic-reoxygenated ventricular strips of the rat heart

The investigation aimed to assess the effects of hypoxic preconditioning in right ventricle strips of fed and 24-h fasted rats, which display a fast fatty acid catabolism, and to ascertain whether these effects are associated with changes in the tissue levels of long-chain acylCoA and acyl carnitine and glycolytic activity. Strips were mounted isometrically in Krebs-bicarbonate solution with 10 mM dextrose and paced at 1 Hz. Strips were exposed to 30 min hypoxia and 60 min reoxygenation with or without a previous preconditioning cycle of 5 min hypoxia followed by a 10 min reoxygenation. During hypoxia the fasted rat strips underwent a greater contracture with respect to the fed group. Preconditioning reduced the contracture strength and accelerated the post-hypoxic recovery only in the fasted rat strips. Hypoxia evoked an increase in the acylCoA and acyl carnitine tissue-contents of the strips which reached higher levels in the fasted than in the fed rat groups. Preconditioning had no effects on the content of these metabolites. During hypoxia lactate output was lower in the fasted than in the fed rat strips and preconditioning abolished this decrease. These data suggest that the protective effects of hypoxic preconditioning occur in the heart tissue predisposed to the oxidation of fatty acid and can not be ascribed to changes in the accumulation of acylCoA and acyl carnitine but could be due, at least in part, to an activation of glycolysis.     

Changes in tissue levels of carnitine and other metabolites during myocardial ischemia and anoxia

The induction of ischemia in the open chest dog, or anoxia in the perfused rat heart, causes dramatic changes in the tissue levels of free acyl carnitine and related metabolites. During the early phase of ischemia or anoxia the tissue levels of free carnitine decline, while acetyl carnitine rapidly increases. These changes are accompanied by elevation in long-chain acyl carnitine, long-chain acyl CoA, and lactate and by decreases in acetyl CoA, CoA, ATP, and creatine phosphate. As the degree of ischemia becomes more severe, carnitine appears to be lost from the myocardium. A scheme is presented which relates carnitine-linked mitochondrial metabolism to the activity of carnitine acyl transferase, ANT, carnitine/acyl carnitine translocase, creatine phosphokinase, and pyruvate dehydrogenase. It is suggested that the conversion of carnitine to acyl carnitine during the onset of ischemia may play an important role, by virtue of its effect on these enzymes, in the regulation of metabolism during the early or reversible phase of ischemia.     

Carnitine acyltransferases and their influence on CoA pools in health and disease

Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed.     

The properties and regulation of pantothenate kinase from rat heart

Pantothenate kinase (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33), the first enzyme in the pathway of CoA synthesis, was partially purified from rat heart. A study of the properties of the kinase showed that it possesses a broad pH optimum between 6 and 9, is activated or inhibited nonspecifically by various anions, and has MgATP as the nucleotide substrate. The Km for MgATP is 0.6 mM and that for pantothenate is 18 microM. CoA and acyl esters of CoA are inhibitors of the kinase with the inhibition by acetyl-CoA being only slightly greater than that by free CoA. The inhibition by free CoA is uncompetitive with respect to pantothenate concentration, with a Ki for inhibition of 0.2 microM. L-Carnitine was found to be a nonessential activator of the kinase. This compound had no effect by itself but specifically reversed the inhibition of the kinase by CoA. The Ka for deinhibition by L-carnitine is 0.27 mM. Free carnitine content was measured in perfused hearts and is found to vary in correlation with perfusion conditions that are known to alter rates of intracellular phosphorylation of pantothenate. These properties of pantothenate kinase provide a potential mechanism for the control of CoA synthesis. The enzyme is regulated by feedback inhibition by CoA and its acyl esters and this inhibition is modified by changes in the concentration of free carnitine.     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

Malonyl coenzyme A affects insulin-stimulated glucose transport in myotubes

There seems to be an association between increased concentrations of malonyl coenzyme A (malonyl CoA) in skeletal muscle and diabetes and/or insulin resistance. The purpose of the current study was to test the hypothesis that treatments designed to manipulate malonyl CoA concentrations would affect insulin-stimulated glucose transport in cultured C2C12 myotubes. We assessed glucose transport after polyamine-mediated delivery of malonyl CoA to myotubes, after incubation with dichloroacetate (which reportedly increases malonyl CoA levels), or after exposure of myotubes to 2-bromopalmitate, a carnitine palmitoyl transferase I inhibitor. All three of these treatments prevented stimulation of glucose transport by insulin. We also assayed glucose transport after 30 min of inhibition of acetyl coenzyme A carboxylase (ACC), the enzyme which catalyzes the production of malonyl CoA. Three unrelated ACC inhibitors (diclofop, clethodim, and Pfizer CP-640186) all enhanced insulin-stimulated glucose transport. However, none of the treatments designed to manipulate malonyl CoA concentrations altered markers of proximal insulin signaling through Akt. The findings support the hypothesis that acute changes in malonyl CoA concentrations affect insulin action in muscle cells but suggest that the effects do not involve alterations in proximal insulin signaling.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.