Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat.

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.     

Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.     

Differentiation of rapid and slower-acting effects of insulin on mitochondrial processes in brown adipose tissue from streptozotocin-diabetic rats.

Insulin treatment of streptozotocin-diabetic rats restores the depressed palmitoyl-group oxidation observed in brown-adipose-tissue mitochondria from diabetic rats. A relatively rapid effect of insulin (5 h) to increase carnitine-dependent oxidation of palmitoyl-CoA and to increase overt carnitine palmitoyltransferase activity is differentiated from a slower effect of the hormone (1 day) to increase palmitoylcarnitine oxidation.     

The localization of palmitoyl-CoA: Carnitine palmitoyltransferase in rat liver

1. The distribution of palmitoyl-CoA:carnitine palmitoyltransferase has been studied in subcellular fractions of rat liver. By using two different estimations for the enzyme activity and by differential centrifugation and linear sucrose density gradient centrifugation, the enzyme is shown to be localized both in mitochondria and microsomes.

2. The mitochondrial palmitoyl-CoA: carnitine palmitoyltransferase is localized in the inner membrane plus matrix fraction.

3. During palmitate oxidation by isolated mitochondria, in the presence of a physiological concentration of carnitine, palmitoylcarnitine accumulates. From this and experiments with sonicated mitochondria, it is concluded that the capacities of long-chain fatty acid activation and of palmitoyl-CoA:carnitine palmitoyltransferase in vitro by far exceed the capacity of fatty acid oxidation.     

Palmitate oxidation by the mitochondria from volume-overloaded rat hearts

In this work, an attempt was made to identify the reasons of impaired long-chain fatty acid utilization that waspreviously described in volume-overloaded rat hearts. The most significant data are the following: (1) The slowing down of long-chain fatty acid oxidation in severely hypertrophied hearts cannot be related to a feedback inhibition of carnitine palmitoyltransferase I from an excessive stimulation of glucose oxidation since, because of decreased tissue levels of L-carnitine, glucose oxidation also declines in volume-overloaded hearts. (2) While, in control hearts, the estimated intracellular concentrations of free carnitine are in the range of the respective K_m of mitochondrial CPT I, a kinetic limitation of this enzyme could occur in hypertrophied hearts due to a 40% decrease in free carnitine. (3) However, the impaired palmitate oxidation persists upon the isolation of the mitochondria from these hearts even in presence of saturating concentrations of L-carnitine. In contrast, the rates of the conversion of both palmitoyl-CoA and palmitoylcarnitine into acetyl-CoA are unchanged. (4) The kinetic analyses of palmitoyl-CoA synthase and carnitine palmitoyltransferase I reactions do not reveal any differences between the two mitochondrial populations studied. On the other hand, the conversion of palmitate into palmitoylcarnitine proves to be substrate inhibited already at physiological concentrations of exogenous palmitate. The data presented in this work demonstrate that, during the development of a severe cardiac hypertrophy, a fragilization of the mitochondrial outer membrane may occur. The functional integrity of this membrane seems to be further deteriorated by increasing concentrations of free fatty acids which gives rise to an impaired functional cooperation between palmitoyl-CoA synthase and carnitine palmitoyltransferase I. In intact myocardium, the utilization of the generated in situ palmitoyl-CoA can be further slowed down by decreased intracellular concentrations of free carnitine.     

Lipid oxidation by heart mitochondria from young adult and senescent rats

1. State-3 (i.e. ADP-stimulated) rates of O(2) uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O(2) uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[(3)H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O(2) uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O(2) uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O(2) uptake with palmitoylcarnitine in senescence is discussed.     

Effects of DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites.

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.     

The carnitine-independent oxidation of palmitate plus malate by moth flight-muscle mitochondria

Mitochondria isolated from the flight muscle of the southern armyworm moth, Prodenia eridania, can oxidize palmitate+malate very rapidly. Added carnitine had no effect on the rate of oxidation of palmitate+malate by flight-muscle mitochondria from two species of moths, and carnitine palmitoyltransferase could not be detected in Prodenia by direct assay. Palmitoylcarnitine was not oxidized by moth mitochondria, but when added in low concentrations it reversibly suppressed the oxidation of palmitate. The evidence indicates that carnitine is not involved in fatty acid degradation by moth flight muscle. Added thiols, including CoA, also suppressed palmitate+malate oxidation. An ATP-dependent fatty acyl-CoA synthetase is present in moth mitochondria.     

The synthesis of palmitoylcarnitine by etio-chloroplasts of greening barley leaves

CoASH, Mg²⁺, ATP and (-)-carnitine were found to be essential for the production of palmitoylcarnitine from palmitate by purified barley etio-chloroplasts. It was concluded that long-chain acyl CoA synthetase (palmitoyl CoA synthetase, EC 6.2.1.3) and carnitine long-chain acyl-transferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity were present in the etio-chloroplasts. It is suggested that the long-chain acylcarnitine formed may move more easily through membrane barriers than the long-chain acyl CoA compound. Also or alternatively this enzyme may spare CoA by transferring long-chain acyl groups from long-chain acyl CoA to carnitine.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria

1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C(4)-C(16). Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of beta-oxidation after the formation intramitochondrially of its metabolites.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.     

Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.-) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with epsilon(mM) 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg(2+) and corresponded to a previously described ;external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg(2+). Of these two ;internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.-), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.     

Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.     

Inhibition of mitochondrial carnitine palmitoyl transferase by 2-tetradecylglycidic acid (McN-3802) (Preliminary Communication)

The oral hypoglycemic agent, 2-tetradecylglycidic acid (McN-3802), which has been reported to inhibit the oxidation of long chain but not short chain fatty acids in isolated rat hepatocytes and muscle preparations, inhibited the oxidation of palmitoyl CoA and palmitic acid by rat liver mitochondria. The drug itself, which is a fatty acid analog, was not oxidized by mitochondria. Evidence is presented that 2-tetradecylglycidic acid (or its coenzyme A ester) inhibits fatty acid oxidation by irreversibly inhibiting mitochondrial carnitine palmitoyltransferase. The drug did not inhibit mitochondrial palmitoyl-CoA synthetase.     

Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Peroxisomal carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCl from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an Mr 64,000 polypeptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an Mr 64,000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal carnitine palmitoyltransferase, but not with mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. In addition, anti-peroxisomal carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Compared with mitochondrial carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyltransferase was 8.5, differing from that of mitochondrial carnitine palmitoyltransferase. The Km value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 microM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 microM), palmitoyl-L-carnitine (43 microM) and CoA (9 microM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C16) and similar Km values (30-50 microM) for acyl-CoAs, acylcarnitine and CoA, and a constant Km value (approximately 150 microM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long-chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.     

Decrease of fatty acid oxidation, ketogenesis and gluconeogenesis in isolated perfused rat liver by phenylalkyl oxirane carboxylate (B 807-27) due to inhibition of CPT I (EC 2.3.1.21)

Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (B 807-27 or POCA) inhibits ketogenesis from endogenous and exogenous long-chain fatty acids and 14CO2 production from [U-14 C]palmitate, but not from [U-14 C]palmitoylcarnitine or octanoate, and inhibits gluconeogenesis from lactate and pyruvate in perfused livers of starved rats. Inhibition of ketogenesis, 14CO2 production and gluconeogenesis was complete at concentrations of 10 mumol/l POCA, but onset was more rapid for inhibition of ketogenesis and CO2 production than for gluconeogenesis. Infusion of octanoate abolished inhibition of all three processes. Experiments with isolated rat liver mitochondria showed that carnitine palmitoyltransferase I (EC 2.3.1.21) is inhibited by POCA-CoA. The inhibitory process is dependent on time and concentration, and more pronounced in mitochondria from fed than from fasted rats. Concentrations required for 50% inhibition after 20 min preincubation with POCA-CoA are 0.02, 0.06 and 0.1 mumol/l in liver mitochondria from fed, 24-h-fasted and 48-h-fasted rats, respectively. The inhibitor appears to be tightly bound to the enzyme. The extent of inhibition of carnitine palmitoyltransferase I correlates well with the hypoglycaemic and hypoketonaemic effects of the compounds in fasted rats. We conclude that specific inhibition of the enzyme leads, due to inhibition of long-chain fatty acid utilization, to depressed ketogenesis and gluconeogenesis and, in consequence, to hypoglycaemic and hypoketonaemia in vivo under gluconeogenic and ketogenic conditions.     

Inhibition of hepatic fatty acid oxidation by bezafibrate and bezafibroyl CoA

The acute effect of the hypolipidemic agent bezafibrate on fatty acid oxidation was studied in rat hepatocytes and mitochondria. Bezafibrate caused a concentration-related inhibition of oleate oxidation in liver cells. In mitochondria bezafibrate inhibited the oxidation of palmitoyl CoA but had no effect on palmitoylcarnitine oxidation, suggesting the site of inhibition was the formation of the carnitine derivative. Bezafibrate and bezafibroyl CoA inhibited the overt carnitine palmitoyltransferase (I) in rat liver mitochondria with comparable potency but with distinct kinetics. The inhibition caused by bezafibrate was not prevented by omission of Mg++-ATP from the assay mixture, indicating activation of bezafibrate to bezafibroyl CoA was not required for inhibition. The data demonstrate that bezafibrate, like several other peroxisome proliferating agents, inhibits mitochondrial fatty acid oxidation in rat liver. The inhibition may be relevant to the mechanism of peroxisome proliferation.     

Effect of carnitine on mitochondrial oxidation of palmitoylcarnitine

The effects of carnitine on the metabolism of palmitoylcarnitine were studied by using isolated rat liver mitochondria. Particular attention was given to carnitine acyltransferase-mediated interactions between carnitine and the mitochondrial CoA pool. Carnitine concentrations less than 1.25mm resulted in an increased production of acetylcarnitine during palmitoylcarnitine oxidation. Despite this shunting of C₂ units to acetylcarnitine formation, no change was observed in the rate of oxygen consumption or major product formation (citrate or acetoacetate). Further, no changes were observed in the mitochondrial content of acetyl-CoA, total acid-soluble CoA or acid-insoluble acyl-CoA. These observations support the concept, based on studies in vivo, that the carnitine/acylcarnitine pool is metabolically sluggish and the acyl-group flux low as compared with the CoA/acyl-CoA pool. Acid-insoluble acyl-CoA content was decreased and CoA content increased at carnitine concentrations greater than 1.25mm. When [¹⁴C]carnitine was used in the incubations, it was demonstrated that this resulted from acid-insoluble acylcarnitine formation from intramitochondrial acid-insoluble acyl-CoA mediated by carnitine palmitoyltransferase B. Again, the higher carnitine concentrations resulted in no changes in the rates of oxygen consumption or major product formation. The above effects of carnitine were observed whether citrate or acetoacetate was the major product of oxidation. In contrast, an increase in acetyl-CoA concentration was observed at high carnitine concentrations only when acetoacetate was the product. Since the rate of acetoacetate production was not changed, these higher acetyl-CoA concentrations suggest that a new steady state had been established to maintain acetoacetate-production rates. Since there was no change in acetyl-CoA concentration when citrate was the major product, a change in the activity of the pathway utilizing acetyl-CoA for ketone-body synthesis and the potential regulation of this pathway must be considered.     

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.     

Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.