Development of fatty acid oxidation in neonatal guinea-pig liver.

The capacity of foetal and neonatal liver to oxidize short-, medium- and long-chain fatty acids was studied in the guinea pig. Liver mitochondria from foetal and newborn animals were unable to synthesize ketone bodies from octanoate, but octanoylcarnitine and palmitoylcarnitine were readily ketogenic. The ketogenic capacity at 24 h after birth was as high as in adult animals. Hepatocytes isolated from term animals were unable to oxidize fatty acids, but at 6 h after birth production of 14CO2, acid-soluble products and acetoacetate from 1-14C-labelled fatty acids was 40-50% of the rates at 24 h. At 12 h of age these rates had already reached the 24 h values and did not change during suckling in the first week of life. The activities of hepatic fatty acyl-CoA synthetases, which were minimal in the foetus or at term, increased to maximal values in 12-24 h. The data show that the capacity for beta-oxidation and ketogenesis develops maximally in this species during the first 6-12 h after birth, and appears to be partly dependent on the development of fatty acid-activating enzyme.     

In vitro fatty acid oxidation rates in rat heart and liver

Experiments were conducted to test the effects of age, sex, and level and type of dietary fat on the oxidation rates of carboxyl- and uniformly-labeled linoleate, oleate and palmitate. There were no significant differences due to age, sex, nor diet alone but a statistically significant (P < 0.05) interaction between sex and tissue was found. The latter appeared to be due to the slower rate displaced by liver homogenates from male rats than females. CO₂ was more rapidly labeled from carboxyl- than from uniformly-labeled fatty acids. In heart, palmitate was oxidized at a faster rate than linoleate with oleate demonstrating the slowest rate. In liver, the relative rates were linoleate > palmitate ? oleate. Incubation conditions, tissue interactions, position of label and end products recovered are discussed in relation to interpretation of results in studies of fatty acid metabolism.     

Diltiazem inhibits fatty acid oxidation in the isolated perfused rat liver

The effects of diltiazem on fatty acid metabolism were measured in the isolated perfused rat liver and in isolated mitochondria. In the perfused rat liver diltiazem inhibited oxygen uptake and ketogenesis from endogenous substrates. Ketogenesis from exogenously supplied palmitate was also inhibited. The β-hydroxybutyrate/acetoacetate ratio in the presence of palmitate alone was equal to 3·2. When the fatty acid and diltiazem were present simultaneously this ratio was decreased to 0·93, suggesting that, in spite of the inhibition of oxygen uptake, the respiratory chain was not rate limiting for the oxidation of the reducing equivalents coming from β-oxidation. In experiments with isolated mitochondria, incubated in the presence of all intermediates of the Krebs cycle, pyruvate or glutamate, no significant inhibition of oxygen uptake by diltiazem was detected. Inhibition of oxygen uptake in isolated mitochondria was found only when palmitoyl CoA was the source of the reducing equivalents. It was concluded that a direct effect on β-oxidation may be a major cause for the inhibition of oxygen uptake caused by diltiazem in the perfused liver. © 1997 John Wiley & Sons, Ltd.     

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver

Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi­dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O₂ consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.     

Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells.

The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal beta-oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C16 and C18 fatty acids recovered, was observed when 18:2(n-6), 20:3(n-6), 18:3 (n-3) and 20:5(n-3), but not when 20:4(n-6) and 22:4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial acetyl-CoA produced from peroxisomal beta-oxidation is more easily used for fatty acid new synthesis than acetyl-CoA from mitochondrial beta-oxidation. The delta 6 and delta 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.     

Intermediates in fatty acid oxidation

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD⁺, CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O₂ electrode. 2. Label from [1-¹⁴C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-¹⁴C]octanoyl-CoA. 3. Oxidation of [8-¹⁴C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-¹⁴C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.     

Comparison of fatty acid oxidation in mitochondria and peroxisomes from rat liver

Oxygen uptake with succinate or palmitoyl-CoA as substrates can be measured in rat liver mitochondria that have been isolated by sucrose density gradient centrifugation providing the fractions are diluted with a 30 mM phosphate buffer rather than with an isotonic medium. Separate assay procedures were used to measure peroxisomal and mitochondrial β-oxidation of palmitoyl-CoA in the fractions of a sucrose gradient used to separate these organelles. A preliminary estimate of the ratio of palmitoyl-CoA oxidation by the mitochondrial fraction relative to the surviving peroxisomes from livers of male rats was 3.2.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.     

Effect of inhibition of fatty acid oxidation on neonatal liver carnitine content

The hypoglycaemic agent 2-tetradecylglycidic acid (compound McN-3802) caused an increase in total liver carnitine content, this being due primarily to an increase in the free carnitine pool. In the neonatal animal, this may represent a mechanism to overcome the inhibitory effect of fatty acid oxidation by the drug.     

Inhibitors of fatty acid oxidation

This review discusses inhibitors of fatty acid oxidation for which sites and mechanisms of inhibition are reasonably well understood. Included in this review are hypoglycin, an inhibitor of butyryl-CoA dehydrogenase (EC 1.3.99.2), 4-pentenoic acid, 2-bromooctanoic acid, and 4-bromocrotonic acid all of which inhibit mitochondrial thiolases (EC 2.3.1.9 and 2.3.1.16) as well as several inhibitors of carnitine palmitoyltransferase I (EC 2.3.1.21) as for example 2-tetradecylglycidic acid, 2-bromopalmitic acid and aminocarnitine. Most of these inhibitors of fatty acid oxidation have been shown to cause hypoglycemia in animals and some also cause hypoketonemia. The advantages and limitations of using these inhibitors in metabolic studies are discussed.     

Malonyl-CoA content and fatty acid oxidation in rat muscle and liver in vivo

Malonyl-CoA acutely regulates fatty acid oxidation in liver in vivo by inhibiting carnitine palmitoyltransferase. Thus rapid increases in the concentration of malonyl-CoA, accompanied by decreases in long-chain fatty acyl carnitine (LCFA-carnitine) and fatty acid oxidation have been observed in liver of fasted-refed rats. It is less clear that it plays a similar role in skeletal muscle. To examine this question, whole body respiratory quotients (RQ) and the concentrations of malonyl-CoA and LCFA-carnitine in muscle were determined in 48-h-starved rats before and at various times after refeeding. RQ values were 0.82 at baseline and increased to 0.93, 1. 0, 1.05, and 1.09 after 1, 3, 12, and 18 h of refeeding, respectively, suggesting inhibition of fat oxidation in all tissues. The increases in RQ at each time point correlated closely (r = 0.98) with increases (50-250%) in the concentration of malonyl-CoA in soleus and gastrocnemius muscles and decreases in plasma FFA and muscle LCFA-carnitine levels. Similar changes in malonyl-CoA and LCFA-carnitine were observed in liver. The increases in malonyl-CoA in muscle during refeeding were not associated with increases in the assayable activity of acetyl-CoA carboxylase (ACC) or decreases in the activity of malonyl-CoA decarboxylase (MCD). The results suggest that, during refeeding after a fast, decreases in fatty acid oxidation occur rapidly in muscle and are attributable both to decreases in plasma FFA and increases in the concentration of malonyl-CoA. They also suggest that the increase in malonyl-CoA in this situation is not due to changes in the assayable activity of either ACC or MCD or an increase in the cytosolic concentration of citrate.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.