Activation Of Long-chain Fatty Acids Research Articles

Carnitine transport and exogenous palmitate oxidation in chronically volume-overloaded rat hearts

L-Carnitine transport and free fatty acid oxidation have been studied in hearts of rats with 3-month-old aorto-caval fistula. For carnitine transport experiments, the hearts were perfused via the ascending aorta with a bicarbonate buffer containing 11 mM glucose and variable concentrations L-[14C]carnitine (10-200 microM). In some experiments, the active component of carnitine transport was suppressed by the adjunction of 0.05 mM mersalyl acid. The subtraction of passive from total transport allowed reconstruction of the saturation curves of the carrier-mediated transport of L-carnitine. Our data suggest that at a physiological carnitine concentration (50 microM), the rate of [14C]carnitine accumulation was significantly depressed in mechanically overloaded hearts. In addition, according to Lineweaver-Burk analysis, the affinity of the membrane carrier for L-carnitine was considerably diminished (Km carnitine 125 instead of 83 microM, Vmax unchanged). The above alterations of L-carnitine transport did not result from a decrease of the transmembrane gradient of sodium, since the intracellular Na+ content of the hypertrophied hearts was quite similar to that of control hearts. The ability of atrially perfused, working hearts to oxidize the exogenous free fatty acids was assessed from 14CO2 production obtained in the presence of [U-14C]palmitate or [1-14C]octanoate. The total 14CO2 production, expressed per min per g dry weight, was significantly diminished in hearts from rats with the aorto-caval fistula if 1.2 mM palmitate was used. On the other hand, in the presence of 2.4 mM octanoate, a substrate which circumvents the carnitine-acylcarnitine translocase, no such reduction of the 14CO2 production could be detected. Our results suggest that the decrease of L-carnitine transport, resulting in a significant depression of tissue carnitine, may impair long-chain fatty acid activation and/or translocation into mitochondria. In contrast, the oxidation of short-chain fatty acids, the activation of which takes place directly in mitochondrial matrix, is not limited in volume-overloaded hearts.

Kinetics of human spermatozoa long-chain fatty acid: CoASH ligase.

The kinetics of long-chain saturated fatty acid activation were studied in the supernatant obtained from Triton-treated human spermatozoa. Sperm long-chain fatty acid: CoASH ligase (AMP) (E.C. 6.2.1.3) was able to activate myristic, palmitic, and stearic acids, but was incapable of utilizing lauric, arachidic, and behenic acids. Peak activity was obtained with palmitic acid. Although the Kms for each fatty acid were similar (4.3 to 5.0 microM), the Vmax was several-fold higher for palmitate. In contrast, ligase from liver homogenates assayed under identical conditions activated lauric through stearic acids, with maximal rates being noted with myristic acid. When compared with nongerminal tissues, sperm ligase appears unique because of its narrower acyl substrate specificity.

Fatty acid activation and transfer in blood cells of patients with muscular dystrophy

Two membrane-bound enzymes, concerned in repair of erythrocyte membranes, have been investigated in patients with muscular dystrophy. The activation of long-chain fatty acids is normal in erythrocytes from Duchenne patients, but increases twofold in cells from myotonic dystrophy patients (congenital form). This alteration is not present in leucocytes. In all leucocytes tested palmitate was the preferred substrate while palmitoleate and linoleate were activated at a lower rate. In the erythrocytes the 3 fatty acids were activated at the same rate. Carnitine palmitoyltransferase was not significantly altered in erythrocytes of both groups of patients.

Fatty Acid Activation: Specificity, Localization, and Function

Publisher Summary This chapter discusses fatty acid activation. Most cells can metabolize fatty acids. The most important pathways are β-oxidation to acetyl-CoA; esterification with glycerol phosphate, glycerol derivatives, sphingosine, glycol, and long-chain alcohols; chain elongation; desaturation; and α and ω oxidation. Fatty acid molecules are completely ionized at physiological pH values. The carboxylate ion is chemically unreactive toward nucleophilic substitution. Esters, thioesters, and acid anhydrides, in which both the carboxylate resonance and the negative charge on the carboxyl group disappear, are more reactive. Both microsomal and mitochondrial long-chain fatty acid activation is stimulated by the presence of high concentrations of several salts. Both activities are also strongly stimulated by the addition of the particle-free supernatant from several rat tissues. Purified mammalian acyl-CoA synthetases are very specific in their nucleotide requirement.

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

In isolated hepatic mitochondria, sodium acetate had little effect on the oxidation of octanoate, but conspicuously inhibited the oxidation of palmitate. This differential effect of acetate on long-chain and short-chain fatty acid oxidation is not due to inhibition of the activation or transfer of long-chain fatty acids into the mitochondria. Both palmitate and octanoate reduced CO 2 production from acetate. Palmitate and octanoate mutually inhibited CO 2 production from each other to the same extent. Acetate stimulated ketogenesis from palmitoyl-1-carnitine to the same extent as it inhibited oxygen uptake and CO 2 production from palmitate. This suggests that acetate causes a redistribution of the end products of palmitate oxidation toward ketogenesis rather than toward total oxidation to CO 2 and H 2O. Acetyl CoA derived from acetate or palmitate may share a common pool or pathway, thus each is mutally inhibitory toward the oxidation of the other. Either because of the existence of separate pools, or because octanoate is the preferred substrate, acetate metabolism does not inhibit O 2 uptake or CO 2 production from octanoate, whereas the oxidation of octanoate dilutes the CO 2 produced from labeled acetate. This may be explained by compartmentation or preferred pathways for the disposition of acetyl CoA derived from different sources.

Palmitoyl-coenzyme A synthetase. Mechanism of reaction.

The mechanism of long-chain fatty acid activation catalysed by highly purified microsomal palmitoyl-CoA synthetase was investigated. The kinetics of the overall reaction were found to conform to the Bi Uni Uni Bi Ping Pong mechanism. (18)O was transferred from [(18)O]palmitate to AMP and palmitoyl-CoA exclusively. The enzyme intermediate formed appeared to consist of enzyme-bound palmitate; this formation occurred only in the presence of ATP. However, the involvement of palmitoyl-AMP in the reaction catalysed by the purified enzyme has proved difficult to establish.

Microsomal palmitoyl coenzyme A synthetase from rat liver. Partial and exchange reactions.

The partial and exchange reactions of long-chain fatty acid activation were determined by using purified microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3). No significant ATP formation from palmitoyl-AMP and PP(i), nor palmitoyl-AMP-dependent CoA disappearance could be demonstrated. Similarly, no palmitate-dependent [(32)P(2)]PP(i)-ATP exchange was catalysed by the pure enzyme. The above partial and exchange reactions were, however, catalysed by the parent microsomal fraction at a rate similar to that of the overall reaction. The implications of these results are discussed.

Some properties of microsomal fatty acid activating enzyme of rat liver

Some properties of microsomal long-chain fatty acid activating enzyme(s) have been studied by hydroxamate formation method or by following formation of [1- 14C]-acyl-CoA from [1- 14C]fatty acids. Unlike with palmitate, hydroxamate formation with laurate requires the presence of lecithin. Lecithin appears to bind fatty acids reversibly and the detergent-type inhibition of the enzyme by excess of fatty acids is thereby prevented. In lecithin-supplemented medium saturated fatty acids from decanoate to stearate were activated at appreciable rates by microsomes of rat liver as well as by homogenates of heart. Separate activity peaks were seen with laurate and palmitate which suggests that more than one enzyme may be involved in the activation of longchain saturated fatty acids. Rates of activation of hexadecanedioate, 12-hydroxystearate, 12-hydroxyoleate, elaidate and oleate are also given. The acyl-CoA synthetase activity, with palmitate and oleate as substrates, is optimal in the pH range 7.2–8.8. At 37 °C, the enzyme(s) is (are) unstable but this effect is independent of pH in the range 7.0–8.8. Presence of high concentration of potassium phosphate or of 5 mM ATP stabilize the enzyme activity. High concentrations of salt increase the apparent K m values for ATP and CoA, as well as the V of the acyl-CoA synthetase reaction. The enzyme shows high affinity for fatty acids but exact determination of K m value is not possible because of marked unspecific binding of free fatty acids to microsomes.

Inhibition of long-chain fatty acid activation by α-bromopalmitate and phytanate

α-Bromopalmitate inhibits the activation of long-chain fatty acids by rat liver preparations. This inhibitory effect is not related to the amount of protein, is reversible and appears to be of a competitive type. An α- bromo or an α- hydroxy substitution slowed the rate of activation to about 1 20th of that of the corresponding unsubstituted fatty acid. Oxidation of palmitate as well as palmitate incorporation into glycerides by homogenates of heart was also inhibited by α-bromopalmitate. Evidence obtained shows that these inhibitory effects are not related to the possible surfactant property of this long-chain free fatty acid. Thus the reported selective inhibitory effect of α-bromopalmitate on fatty acid oxidation in intact cells may involve inhibition of activation in addition to the known inhibition of carnitine palmitoyltransferase by CoA esters of α-bromo long-chain acids. Activation of palmitate and palmitate oxidation were also inhibited by phytanate. Hence it appears possible that the accumulation of phytanic acid as in Refsum's disease may interfere with the normal oxidation of fatty acids.

Fatty acid activation and acyl transfer in organs from rats in different nutritional states

1. 1. The activities of the fatty acid activating enzymes, carnitine palmityltransferase and glycerophosphate acylating enzyme(s) have been measured in the liver, heart, and epididymal fat from fed, fasted, glucose-refed, and fat-fed rats. 2. 2. Fasting gave no significant change in the activation of long-chain fatty acids, but glucose-refeeding caused a decreased activation rate in the liver. 3. 3. The activation of short- and medium-chain fatty acids was reduced during fasting in all the investigated organs. Glucose-refeeding increased the activation of heptanoate in the liver and all the short- and medium-chain acids in heart. 4. 4. The carnitine palmityltransferase activity in the liver increased both in fasted and fat-fed animals. Glucose-refeeding normalized the activity. No significant changes were found in the other tissues. 5. 5. The glycerophosphate acylation in the liver and heart decreased during fasting and fat-feeding. After glucose-refeeding the activity in the liver increased again to the level found in the controls. 6. 6. It is suggested that in rat liver the activity of the fatty acid-activating enzymes, the carnitine palmityltransferase and the glycerophosphate acylating enzyme(s) participate in the regulation of fatty acid metabolism. The study gives no conclusive evidence for such mechanisms in the heart or epididymal fat.

Dual localization and properties of ATP-dependent long-chain fatty acid activation in rat liver mitochondria and the consequences for fatty acid oxidation

1. Direct evidence is given for the existence of two ATP-dependent palmitoyl-CoA synthesizing enzymes, localized in different compartments of the rat-liver mitochondrion. 2. About 90% of the total activity of rat liver mitochondria is localized in the mitochondrial outer membrane and about 10% in the inner membrane-matrix compartment. 3. The two enzyme systems show different apparent K m 's for fatty acid and ATP, and different apparent K i 's for AMP and adenosine. 4. The inner membrane-matrix enzyme, in contrast to the outer membrane and the microsomal enzyme, is strongly inhibited by octanoate. 5. Comparison of the kinetics of the two ATP-dependent long-chain fatty acid-activating enzymes with the kinetics of long-chain fatty acid oxidation shows, that during fatty acid oxidation at low concentrations of palmitate or oleate, in the presence of carnitine, the outer membrane acyl-CoA synthetase is operating. In the absence of carnitine and at high concentrations of long-chain fatty acids the activation reaction occurs in the inner membrane-matrix compartment of the mitochondrion. 6. The long-chain fatty acid concentration needed for half-maximal velocity of fatty acid oxidation by isolated rat liver mitochondria is about 1 μM in the presence of carnitine and 100–200 μM in the absence of carnitine.

Inhibition of hepatic gluconeogenesis and fatty acid oxidation by pent-4-enoic acid.

ARTICLESInhibition of hepatic gluconeogenesis and fatty acid oxidation by pent-4-enoic acidNB Ruderman, CJ Toews, C Lowy, I Vreeland, and E ShafrirNB Ruderman, CJ Toews, C Lowy, I Vreeland, and E ShafrirPublished Online:01 Jul 1970https://doi.org/10.1152/ajplegacy.1970.219.1.51MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByAliphatic Carboxylic Acids: Unsaturated17 August 2012Aliphatic Carboxylic Acids, Unsaturated1 January 2000Role of endogenous fatty acids in the control of hepatic gluconeogenesisArchives of Biochemistry and Biophysics, Vol. 292, No. 1Synthesis and Hypoglycemic Activity of Benzamidophenyl-alkanoic Acid Derivates: New Inhibitors of GluconeogenesisJournal of Pharmaceutical Sciences, Vol. 71, No. 7Effect of methoxyindole 2-carboxylic acid and 4-pentenoic acid on adipose tissue metabolismBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol. 388, No. 2Inhibitors of Fatty Acid Oxidation and the Pathway of Fatty Acid Biosynthesis from GlucoseAmino acid metabolism in lactic acidosisThe American Journal of Medicine, Vol. 52, No. 4Inhibition of long-chain fatty acid activation by α-bromopalmitate and phytanateBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol. 248, No. 2Inhibition of Hepatic Gluconeogenesis by Pent-4-enoic acid: Role of Redox State, Acetyl-CoA and ATP More from this issue > Volume 219Issue 1July 1970Pages 51-57 Copyright & PermissionsCopyright © 1970 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1970.219.1.51PubMed5424858History Published online 1 July 1970 Published in print 1 July 1970 Metrics

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.

Inhibition of triglyceride synthesis in everted intestinal sacs.

SummaryOptimal conditions for synthesis of triglyceride from oleic acid-1-14C in everted intestinal sacs were determined. Using several experimental conditions, it was found that 2-ethyl-n-caproate inhibited oleic acid incorporation into triglyceride by 25-50%. When inhibition of triglyceride synthesis occurred, there was a concomitant increase in mucosal unesterified fatty acids but no increase in the phospholipid, mono- or diglyceride fractions. The inhibition of glyceride synthesis in the intestinal mucosa by 2-ethyl-n-caproate appears to be at the level of long-chain fatty acid activation.

Activation of long-chain fatty acids by rat-gut mucosa

Activation of long-chain fatty acids by rat-gut mucosa