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PHYSIOLOGICAL REVIEWS Vol. 63, No. 4, October 1983 Prided i?i U.S.A. Carnitine-Metabolism and Functions JON BREMER Institute of Medical Biochemistry, University of Oslo, Oslo, Norway 1420 0031-9333/83 $1.50 Copyright 0 1983 the American Physiological 1421 1422 1422 1424 1424 1424 1424 1425 1425 1426 1426 1428 1428 1429 1430 1430 1431 1431 1431 1432 1433 1433 1434 1435 1439 1439 1439 1440 1441 1442 1442 1442 1443 1444 1445 1445 1445 1447 1447 1448 1449 Society Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 I. Introduction .......................................................... .......................................... II. Occurrence and Distribution III. Biosynthesis .......................................................... ............... A. S-adenosylmethionine-6-N-L-lysine methyltransferase ................................. B. Protein (lysine) methyltransferase .................... C. 6-N-trimethyllysine, 2-oxoglutarate dioxygenase ............................ D. 3-Hydroxy-6-N-trimethyllysine aldolase ........................... E. y-Butyrobetaine aldehyde dehydrogenase ........................ F. y-Butyrobetaine,2-oxoglutarate dioxygenase ..................................... IV. Transport of Carnitine Precursors V. Transportofcarnitine ................................................ A. Liver ............................................................. ......................................... B. Skeletal muscle and heart C. Kidney ............................................................ D. Epididymis ........................................................ E. Brain ............................................................. ........................................................ F. Other cells ............................................ VI. Excretion and Degradation A. Excretion ......................................................... .................................... B. Degradation in microorganisms ............................................. C. Degradationininsects .......................................... D. Degradation in mammals ..................................... VII. Regulation of Carnitine Turnover ........... VIII. Function of Carnitine in Mitochondrial Fatty Acid Oxidation .......................... IX. Function of Carnitine in Acetate Metabolism A. Fatty acid synthesis ............................................... .................................. B. Acetate and acetyl-CoA oxidation ............. X. Function of Carnitine in Peroxisomal Fatty Acid Oxidation ................................ XI. How Many Carnitine Acyltransferases? ........................................... XII. Carnitine Acetyltransferase ........................................... A. Isolation and properties ............................................ B. Kinetics and specificity ................................ C. Inhibitors and “suicidal” substrates ......................................... XIII. Carnitine Octanoyltransferase ........................................ XIV. Carnitine Palmitoyltransferase ......................................... A. Purification and properties ............................................ B. Kinetics and specificity C. Effectofdetergents ............................................... ............................. D. Properties in mitochondrial membrane E. Inhibitors ......................................................... ................................................. XV. CarnitineTranslocase October 1983 XVI. XVII. XVIII. XXIII. I. AND FUNCTIONS A. Propertiesand kinetics ............................................ B. Inhibitors ......................................................... Acylcarnitine Hydrolase .............................................. Regulation of Fatty Acid Oxidation in Liver ........................... A. Effect of metabolites and cofactors ................................. B. Changes in mitochondria .......................................... C. Allover regulation ................................................. Fatty Acid Oxidation in Extrahepatic Tissues .......................... A. Heart and skeletal muscle ......................................... B. Brown adipose tissue .............................................. Carnitine Acyltransferases in Newborns ............................... Carnitine and Fertility ................................................ Carnitine and Branched-Chain Amino Acid Metabolism ................ Carnitine in Pathogenic Mechanisms .................................. A. Loss and lack of carnitine ......................................... B. Inborn errors of carnitine metabolism .............................. Summary ............................................................ 1421 1449 1449 1450 1450 1450 1453 1456 1456 1457 1458 1459 1460 1461 1463 1463 1464 1466 INTRODUCTION Carnitine has a long history in biochemistry. It was discovered to be a quantitatively important compound in muscle tissue in 1905 (El), and its chemical structure was determined in 1927 (404). Although the chemical similarity between carnitine and choline inspired extensive physiological and pharmacological studies in the 193Os, such studies did not reveal any definite evidence either for the physiological role of carnitine or for its biosynthesis or degradation. In 1952 Carter et al. (89) created new interest in carnitine when they established that it is a growth factor for the meal worm Tenebrio moZitcw [hence carnitine’s other name, vitamin BT (T for Tenebrio)]. Subsequent studies showed that carnitine-deficient larvae died “fat” when they were starved; that is, they were unable to utilize their fat stores in order to survive (151). The recent steadily growing interest in the function of carnitine has its origin in two papers published in 1955. At that time Friedman and Fraenkel (153) discovered that carnitine can be reversibly acetylated by acetyl coenzyme A (acetyl-CoA), and Fritz (155) showed that carnitine stimulates fatty acid oxidation in liver homogenates. These studies led to the discovery that carnitine carries activated fatty acids across the mitochondrial membrane. The present review deals mainly with carnitine research since 1955. The early literature has been extensively covered in reviews by Fraenkel and Friedman (151, 154) and by Fritz (156). Two symposia on carnitine have been published in separate books (152, 430), and reviews of carnitine palmitoyltransferase (EC 2.3.1.21; 196) and of carnitine metabolism in humans (279-281) have also appeared. Analytical and experimental procedures are not discussed in this review. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 XIX. XX. XXI. XXII. CARNITINE-METABOLISM 1422 II. OCCURRENCE JON AND BREMER Vdume 63 DISTRIBUTION III. BIOSYNTHESIS The ability to synthesize carnitine seems to be nearly as ubiquitous as carnitine itself. Only a few insect larvae of the tenebrionidae family require carnitine (i.e., vitamin BT) in their diets (89, 154). The first convincing evidence for carnitine biosynthesis in animals was obtained from chick embryos, which contained significant amounts of carnitine, whereas none was found in eggs. When grown on a carnitine-free synthetic medium, the microorganism Neurospora crassa also contained carnitine (150). The first clues to the mechanism of carnitine biosynthesis were obtained in 1961, when it was shown that the methyl groups of carnitine come from methionine (but not from choline; 59,431) and that y-butyrobetaine (but not y-aminobutyric acid or y-dimethylaminobutyrate) is converted to carnitine (60, 242, 243). These results, however, left the origin of the carnitine fourcarbon chain unknown. The origin of this chain was established 10 years later when several investigators showed that labeled lysine is converted to carnitine in N. crassa (201,202) and in the rat (397), with 6-N-trimethyllysine as an intermediate (127, 128, 397). Figure 1 shows the conversion of lysine to carnitine. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Carnitine most likely is present in all animal species, in many microorganisms, and in many plants (150, 279, 326). A similar, nearly ubiquitous distribution of carnitine palmitoyltransferase has been found (129,304). The flight muscle of the blowfly (Phormia regina) is an exception: it has a high concentration of carnitine but no detectable carnitine palmitoyltransferase, although a high activity of carnitine acetyltransferase (EC 2.3.1.7) is found (109). The general occurrence of carnitine and of its accompanying carnitine acyltransferases shows that it must have been developed at a phylogenetically early stage, probably during a time closely associated with the development of mitochondria. The concentration of carnitine in different species and in different tissues varies over a wide range. The highest concentrations reported have been found in horseshoe crab muscle (152) and in rat epididymal fluid, in which carnitine can reach a concentration of 60 mM (80). In mammalian tissues the concentration usually varies between 0.1 and a few millimoles per liter (with the highest concentrations in heart and skeletal muscle), and there are relatively great interspecies variations. The carnitine concentration is -1 mM in rat skeletal muscle (81), is -3 mM in human muscle (97), and may be up to 15 mM in ruminant muscle (376, 377). When carnitine is found in plants, the concentration is only a few micromoles per liter (326). October 1983 (Protein) I ysine AND IkH 33) I CH2 I CH2 I II CH kH 331 I CH2 I CH2 I III CH2 - CH2 I CHNH, I COOH CHOH I CHNH2 FH2NH2 COOH COOH I2 (Protein) 6-N-trimethyllysine - I I i(CH3)3 I CH2 I CH2 I CH I2 CHO + 3-Hydroxy- If-Butyrobetaine 6-N-trimethyk lysine aldehyde + glycine 1423 FUNCTIONS IV - i(CH331 I CH2 I CH I 2 CH2 I COOH 8-Butyrobetaine i(CH3)3 I CH2 I CHOH I CH2 I COOH I-ICarnitine FIG. 1. Enzyme steps in the biosynthesis of carnitine. I: S-adenosyl-6-N-L-lysine methyltransferase (yeast) or protein (lysine) methyltransferase (animal tissues). II: 6JWtrimethyllysine, 2-oxoglutarate dioxygenase in animal tissues trimethyllysine must first be liberated from methylated proteins by peptidases. III: 3-hydroxy-6-N-trimethyllysine aldolase. IV: butyrobetaine aldehyde dehydrogenase. V: y-butyrobetaine,%oxoglutarate dioxygenase. In N. crassa free lysine is methylated with S-adenosylmethionine as the methyl donor (55, 340). In animals, however, 6-N-trimethyllysine is formed by the methylation of lysine residues in proteins such as myosin, actin, and histones (321). Carnitine is formed from this 6-N-trimethyllysine after its liberation in protein breakdown, presumably in the lysosomes (139, 235). In the next step the trimethyllysine is hydroxylated to 3-hydroxy-6-Ntrimethyllysine (197,203,223,308,350). The hydroxytrimethyllysine is cleaved to butyrobetaine aldehyde and glycine (193,203). The butyrobetaine aldehyde is then oxidized to butyrobetaine (204), which is finally hydroxylated to carnitine (243, 244). Most animal tissues, including brain and skeletal muscle, contain the enzymes necessary to convert trimethyllysine to butyrobetaine (128, 178, 342, 398). However, the last enzyme [butyrobetaine hydroxylase; also called y=butyrobetaine,2=oxoglutarate dioxygenase (EC 1.14.11.1)] is present only in few tissues, and it also shows species variations in tissue distribution. In all species it is found in the liver. In the rat it is also found to a small extent in the testis, but it is absent from all other tissues (38,95, 128,178). It is present in the kidneys in hamster, rabbit, rhesus monkey, and cat but not in the kidneys of dog, guinea pig, mouse, or rat (145). In humans the enzyme is present in liver, kidney, and brain (144, 342). In liver the conversion of trimethyllysine to butyrobetaine seems to take place in both parenchymal and nonparenchymal cells, whereas the hydroxylation of butyrobetaine to carnitine occurs only in parenchymal cells (139). Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 NH2 I CH2 I CH2 I I CH2 I CH2 I CHNH, I COOH CARNITINE-METABOLISM 1424 JON BREMER A. S-Adenosylmethionine-6-N-h-Lysine Volume 63 Methyltransferase In N. crassa S-adenosylmethionine-6-N-L-lysine methyltransferase (EC 2.1.1.-) is a soluble enzyme with a molecular weight of -22,000. It contains only one peptide chain. The same enzyme transfers all three methyl groups. The second methyl group is transferred about five times faster than the first, and the third methyl group is transferred about three times faster than the second. Thus any accumulation of partially methylated lysine is prevented. No cofactor or prosthetic group for this enzyme has been found (55). (Lysine) Methyltransferase Protein (lysine) methyltransferase (EC 2.1.1.43) is present in all tissues of the rat. It is localized in the nuclei. The Michaelis-Menten constant (K,) for S-adenosylmethionine of the nuclear enzyme is -3 PM, a value well below the intracellular concentrations of S-adenosylmethionine. Partially purified enzyme from calf thymus methylates histone and a variety of proteins. It is not known whether more than one enzyme is active in protein lysine methylation. Mono-, di-, and trimethylated lysines are all found in different proteins (321), but only the trimethyllysine is converted to carnitine (235). The same derivatives have been isolated from human urine (220). Thus only part of the liberated trimethyllysine is converted to carnitine. C’ 6-N-Trimethyllysine, %Oxoglutarate Dioxygenase This enzyme (EC 1.14.11.-) is a dioxygenase localized in the mitochondria of kidney, liver, heart, skeletal muscle, and probably other tissues (389). The enzyme requires a-ketoglutarate and oxygen as cosubstrates and ferrous ions and ascorbic acid for optimal activity (203). The enzyme presumably has a reaction mechanism similar to that of y-butyrobetaine,Z-oxoglutarate dioxygenase and other cy-ketoglutarate-requiring hydroxylases (see sect. 1118’). This is the only enzyme of this type shown to be present in mitochondria. The hydroxylation in isolated mitochondria is stimulated by Ca2+, but this may be an effect on the substrate uptake in the mitochondria (350). So far the enzyme has not been purified. In accordance with the requirement for ascorbic acid, scorbutic guinea pigs show a decreased capacity to convert trimethyllysine and butyrobetaine to carnitine and have less carnitine than normal in heart and skeletal muscle, although the carnitine content in liver and plasma is normal (293). D. 3-Hydroxy-6-N-Triwwthyllysine Aldolase This enzyme cleaves its substrate to butyrobetaine aldehyde and glycine. 3-Hydroxy-6-N-trimethyllysine aldolase may be identical to serine hydrox- Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 B. Protein October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1425 ymethyltransferase (EC 2.1.2.1), because this enzyme catalyzes the reaction (203). Nevertheless the existence of a separate enzyme for the cleavage of 3-hydroxy-6-N-trimethyllysine has not been excluded. In human tissues the highest activity is found in liver (342). The enzyme is inhibited by l-amino-D-proline, an agonist of vitamin Be, leading to accumulation of 3-hydroxy-6-N-trimethyllysine in the cell (138). E. T-Butyrobetaine Aldehyde Dehydrogenase F. y-Butyrobetaine,Z-Oxoglutarate Dioxygenase This enzyme was the first enzyme of carnitine biosynthesis to be studied. The enzyme requires ar-ketoglutarate and oxygen as cosubstrates and ferrous ions and a reducing agent for its activity. Ascorbic acid is most efficient as the reducing agent. The enzyme catalyzes the reaction butyrobetaine + a-ketoglutarate + O2 - carnitine + succinate + CO2 The ferrous ions are assumed to activate oxygen in the reaction, and ascorbic acid most likely protects the ferrous ions and enzyme sulfhydryl (-SH) groups from oxidation. An activating effect of catalase (EC 1.11.1.6) in vitro is probably explained by the removal of inactivating hydrogen peroxide formed by spontaneous oxidation of ascorbic acid and ferrous ions (243). With an ‘*02 atmosphere [‘*O]oxygen is incorporated into both carnitine and succinate. It has therefore been suggested that formation of a peroxide bridge between butyrobetaine and a-ketoglutarate initiates the reaction (241). The enzyme thus is a dioxygenase, incorporating one oxygen in each of the products. Several similar dioxygenases are known (244). The purified enzyme of calf liver is a dimer with two 46,000-dalton subunits. The K, values for butyrobetaine and a-ketoglutarate are 0.5 and 0.8 mM, respectively (231). A similar inducible enzyme has been isolated from a Pseudormmas strain able to grow on butyrobetaine as the sole carbon source (245, 246). Recently it has been found that y-butyrobetaine,2-oxoglutarate dioxygenase catalyzes the decarboxylation of a-ketoglutarate in the presence of Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 This enzyme has been purified from the cytosol of bovine liver. It shows a high specificity for butyrobetaine aldehyde and nicotine adenine dinucleotide (NAD). The &, for butyrobetaine is -4 PM, and the molecular weight of the enzyme has been determined to be 160,000, with subunits of -55,000 daltons (204). In human tissues the highest activity is found in liver and kidney (342). 1426 JON BREMER Vobne 63 both (+)- and (-)carnitine but with no conversion of the carnitine. Hence the enzyme is “uncoupled” by one of its reaction products (195). It is still unknown whether this reaction has any regulatory function in carnitine biosynthesis. IV. TRANSPORT OF CARNITINE PRECURSORS V. TRANSPORT OF CARNITINE Several in vivo studies have appeared on the uptake and exchange of carnitine in different tissues (81,96,441). Because most tissues have a carnitine concentration that is >lO-fold higher than that of blood plasma, an active uptake of carnitine must take place; this uptake, however, must occur with widely varying rates. Brooks and McIntosh (81) have shown that the turnover times for carnitine in kidney, liver, heart, skeletal muscle, and brain in the and 220 h, respectively. Tissue distribution and uptake rat are 0.4,1.3,21,105, are at least partially controlled by hormones, particularly in the liver and epididymis. Fasting or diabetes increase the liver concentration of carnitine (272). This increase is particularly great in sheep, where the liver carnitine increases 7- to 300fold in diabetic animals (377). In the kidneys the concentration is about doubled, whereas heart and skeletal muscle show no change. In rats the implantation of a tumor producing great amounts of prolactin, growth hormone (somatotropin), and adrenocorticotropic hormone (ACTH) increased the liver carnitine nearly lo-fold. At the same time cardiac carnitine dropped (327). Injection of glucagon increases liver carnitine in rats (272), and glucagon also stimulates carnitine uptake in isolated hepatocytes (114). In humans glucagon reduces plasma carnitine (167), presumably because liver uptake is stimulated. Choline-deficient rats have a lowered liver carnitine that is rapidly replenished when choline is given (88). Because choline is not a carnitine precursor (59), it must therefore influence carnitine uptake in the liver by an unknown mechanism. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The formation of 6-N-trimethyllysine and its conversion to butyrobetaine take place in most tissues, but the hydroxylation of butyrobetaine to carnitine occurs only in the liver (and in some species in the kidney). Therefore interorgan transport of butyrobetaine and carnitine takes place. Intestinal digestion of proteins will also liberate trimethyllysine, which can be absorbed. The trimethyllysine is partially converted to butyrobetaine in the intestinal wall. Circulating trimethyllysine is not readily taken up by the liver, but it is taken up and converted to butyrobetaine by the kidneys (444). Nothing is known about the release of butyrobetaine from the tissues. Circulating butyrobetaine is taken up by the perfused liver but is poorly absorbed by perfused kidneys (444). October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1427 n ‘p 0 v -x 15E A -53 % ‘E lon u0 2.> G : - 50 2 I 5e--4 II III trace -1 7% 1 B 4 -16% trace 20 Fraction trace 40 60 no. FIG. 2. Metabolism of r3H]CH3(+) carnitine (A) and PH]CH3(-)carnitine (B) in rat. Different rats (weighing -250 g) were injected subcutaneously with 2 radioactive isomers (~5 pmol), and urine was collected for 24 h. After treatment with alkali to hydrolyze acylcarnitines, the urine was evaporated; the residue was extracted with ethanol, and the extract chromatographed on a column of Dowex 50 H, 200-400 mesh, 1 X 30 cm, with 1.5 N HCl as eluent. Fractions of 5 ml were collected. Percentage of injected radioactivity recovered in different chromatographic peaks is given. I: carnitine; II: methylcholine (?); and III: trimethylaminoacetone. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Sex hormones may also influence carnitine distribution. Female rats have been reported to have a significantly higher liver/plasma carnitine concentration ratio than male rats (52), and the high carnitine uptake and concentration in the epididymis depend on an androgen-induced transport mechanism (42). As Smoswell and Henderson (376) have pointed out, the carnitine content of skeletal muscles in sheep appears to be inversely related to the coenzyme A (CoA) content. Such a relation is not found in liver, however, where both carnitine (272) and CoA (374) increase in fasting animals. The great differences in carnitine uptake in different tissues have been confirmed in studies on isolated organs or cells. Differences in kinetics and stereospecificity have also been observed. (+)Carnitine is eliminated from the body more quickly than (-)carnitine (Fig. 2). Nevertheless even after 24 h a significant fraction of (+)carnitine is retained in different tissues, apparently with a distribution that agrees with the variable stereospecificity of carnitine uptake in the tissues (Table 1). Carnitine is also released by tissues, particularly by liver, gut, and kidneys, which furnish other tissues with carnitine after synthesis, absorption, and reabsorption, respectively. 1428 JONBREMER TABLE 1. Relative 68 uptake of labeled (-#-)- and (-)carnitine Tissu6 (-)Carnitine in tissues injection; 3.2 8.1 19 7.7 31 54 2.4 0.9 (blood -70% = 1).of rH]CH3(-)of (+)carnitine (+)Carnitine 3.6 6.6 8.6 4.8 9 9.5 1.1 0.8 and rH]CH3( +)carnitine (-5 pmol) and 16% of (-)carnitine were recovered A. Liver Isolated liver cells have the ability to accumulate both butyrobetaine and carnitine (116). The maximum rate of carnitine uptake (2.4 nmol. mg-’ protein emin-‘) was found to be about twice that of butyrobetaine. The K, for carnitine uptake (“5 mM), however, was 10 times higher than for butyrobetaine (0.5 mM). Mutual competitive studies and the effect of structural analogues suggest that both compounds are transported by the same carrier, and the lower K, for butyrobetaine shows that this compound is the preferred “substrate.” The liver carrier shows no or almost no stereospecificity, (+)carnitine being taken up about as fast as (-)carnitine. This lack of stereospecificity has been confirmed in vivo (see Table 1). With butyrobetaine the concentration in the cells reached ~100 times that in the medium, whereas with carnitine an accumulation with a factor of -10 was reached. This accumulation of carnitine is about the same as that in the intact animal. Liver cells incubated in a carnitine-free medium release both carnitine and acetylcarnitine. The release of acetylcarnitine is even faster than the release of free carnitine. This release of both carnitine and acetylcarnitine can also be seen in intact rats injected with labeled butyrobetaine (116). A significant stimulation of carnitine uptake by glucagon in isolated liver cells has also been observed (114). These results suggest that the variable acetylcarnitine/carnitine ratios in blood plasma and urine (57, 162, 166) reflect corresponding acetylcarnitine/carnitine ratios in liver, ratios modified by the transport specificities of liver and kidneys. B. Skeletal Muscle and Heart Carnitine uptake in muscle has been studied in heart cells in tissue culture (41,284,285,287,288), in heart myocytes (9), and in isolated skeletal muscle (339, 425). Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Liver Kidney Heart Skeletal muscle Caput epididymidis Cauda epididymidis Testis Brain Relative uptake 24 h after subcutaneous in urine (Fig. 1). Vdume October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1429 C. Kidney Kidneys show the fastest rate of carnitine turnover in the body (81), presumably because of efficient reabsorption from the glomerular filtrate, and kidney slices absorb carnitine from the medium. The Km for (-)carnitine Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The properties of the system for active uptake of carnitine are rather different from those of the liver system. The Km values reported for (-)carnitine vary for different muscle preparations, but all are much lower than the Km found with liver cells (“5 PM in tissue culture heart cells, 60 PM in heart myocytes, and 0.06-0.5 mM in isolated muscles, vs. 5 mM in liver cells). The maximum uptake rate may’ also be only l/l,ooo of the maximum rate in liver. This agrees quite well with the turnover time, which was 100-200 times longer for muscle carnitine than for liver carnitine in vivo (81). The muscle system also is more specific for (-)carnitine, because the Km is significantly higher and the uptake rate significantly lower with both (+)carnitine and butyrobetaine. This stereospecificity has been confirmed in vivo (Table 1). In heart cell cultures the capacity for carnitine uptake increased when cells were grown in the presence of carnitine and when cells were grown in the presence of prednisolone. These effects seemed to be additive (285) and may be why patients with lipid-storage myopathy due to low muscle carnitine levels showed improvement on treatment with carnitine and prednisone (412). Carnitine is also slowly released from muscle cells, and carnitine and carnitine analogues stimulate this release. Apparently release is not simply a reversal of uptake (284). In this connection it is interesting that (+)carnitine injections in rats provoke a carnitine deficiency in heart and skeletal muscle but not in the liver (332) and that (+)carnitine [but not (-)carnitine] can cause muscle weakness (myasthenia) in patients treated with carnitine (18). Recently, Cantrell and Borum (85) have extracted from heart a carnitinebinding protein with properties that agree closely with the carnitine-uptake characteristics of the heart. The highest binding capacity was found in a fraction rich in plasma membrane. It is likely therefore that this protein represents the carrier that transports carnitine across the cell membrane, a carrier different from the carnitine translocase in the mitochondria (see sect. XV). A peculiar feature of this protein is the slow but relatively specific binding of (-)carnitine. Maximum binding was obtained after incubation for 1 h at 25OC. The relatively slow uptake of carnitine in heart may be connected with this slow binding. It is also striking that butyrobetaine is a relatively weak inhibitor of carnitine binding to this heart protein. In liver cells butyrobetaine is taken up with a lower Km than is carnitine (116). It therefore seems likely that the carnitine carrier in the hepatocyte cell membrane is different from that of muscle tissues. 1430 JON BREMER l-+&me 63 uptake in kidney slices was found to be -90 PM; the maximum uptake rate fluid, which is close to the calculated was 22 nmol min-’ ml-’ intracellular normal rate of carnitine reabsorption in the kidneys. The system shows only a moderate stereospecificity, because the Km and the maximum rate for (+)carnitine were 166 PM and 14 nmol min-’ ml-l fluid, respectively (206). No studies have been performed on the renal clearance of (+)carnitine, and thus it is not known whether these results agree with the more rapid elimination of (+)carnitine from the body (Fig. 1). Evidently the kidneys must also possess a system for the release of carnitine into the bloodstream. A relatively rapid “loss” of carnitine from kidney slices was observed, but the nature of this release has not been studied. When (-)carnitine and (+)carnitine were administered separately to rats, 47% of the radioactive (+)carnitine was recovered in the urine after 4 h and 71% was recovered after 24 h, whereas with (-)carnitine ~10% and 16% were recovered in the urine after 4 and 24 h, respectively (Fig. 2). These results agree quite well with the excretion of (+)carnitine (247). Although it is impossible from this experiment to decide exactly to what extent the kidneys selectively clear (+)carnitine from the blood, the results suggest a stereospecific reabsorption of (-)carnitine in the kidneys. A significant uptake of (+)carnitine into the tissues does take place, however, and may explain why (f)carnitine has some toxic effects (18, 332). l l l l Marquis and Fritz (265) were the first to observe the extremely high concentrations of carnitine in the cauda epididymidis and the epididymal fluid and to determine that this high concentration depends on the action of androgen hormones. In the epididymis the androgens induce a carnitinetransport system with the ability to concentrate carnitine in the epididymal lumen (42). In rats this concentration can reach 60 mM, or 2,000 times the concentration in blood (80, 191). As in the kidney, a double transport mechanism must be assumed, because the carnitine passes through the epididymal cells into the lumen. The main concentration gradient is across the brush border at the luminal side of the cell, and androgens seem to induce this transport from the cell into the lumen (45). This assumption of a double transport system is supported by the observation that carnitine uptake in dispersed cells is nonstereospecific, with a K, of ~1 mM for both (+)- and (-)carnitine (213). The overall process is specific for (-)carnitine (440), however, and this is confirmed by in vivo experiments (see Table 1). E. Brain Although carnitine uptake in the brain is extremely slow in vivo @I), brain slices show a relatively rapid uptake (205). The K, for (-)carnitine Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 D. Epididymis October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1431 F. Other Cells Carnitine uptake has been studied in fetal lung fibroblasts and in smooth muscle cells from the aorta (87). The Km for uptake was found to be 6-8 PM [i.e., similar to that found in heart cells in tissue culture (41)]. In Pseudowmnas ueruginosa an active uptake mechanism stereospecific for (-)carnitine is induced when the cells are grown in a carnitine-containing medium (7,229). The induction of this transport system, which also catalyzes a carnitine/carnitine exchange, is inhibited by chloramphenicol, suggesting that a protein carrier is synthesized. An Na+-K+-ATPase also seems to be involved in the active transport. The Km for the carnitine of this system was found to be 0.7 mM. VI. EXCRETION AND DEGRADATION A. Excretion In normal animals carnitine is lost mainly by excretion in the urine. In normal rats 1-2 pmol of carnitine are excreted per day per 100 g of body weight (96, 406). This represents l/15-?&-, of the total body pool and is in agreement with the slow turnover in quantitatively important tissues, such as skeletal muscle (81). In one turnover study, however, the loss of carnitine in rat was estimated to be about three times the amount recovered in the urine. No explanation was found for this discrepancy (81). (56). The In rats the renal clearance of carnitine is co.05 liters/day glomerular filtration rate is ~1.7 liters. day-’ 100 g-l body wt (17). Thus >95% of the carnitine in the glomerular filtrate is reabsorbed. This is similar to amino acid reabsorption in the kidneys. In fasting rats both the plasma concentration and the renal excretion drop at first but then increase above the initial values after fasting for 3-4 days. Both free carnitine and acylcarnitines are excreted (56). l Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 was ~3 mM, whereas (+)carnitine was taken up with a Km of ~10 mM. Thus the affinity of the transporter in brain is similar to that in liver, but the brain shows a more pronounced stereospecificity than the liver, which takes up (-)- and (+)carnitine at about the same rate (116). All other tissues investigated have lower Km values (5 PM-0.5 mM). The brain system is strongly inhibited by y-aminobutyric acid [inhibitor constant (Ki) = 0.6 mM], but this compound has no inhibitory effect in kidney and muscle (147, 205, 339). The discrepancy between the rates of carnitine uptake in vivo and in vitro suggests that the blood-brain barrier limits the access of carnitine to the brain. The nature of the transport across this barrier is unknown. 1432 JON vdurne BREMER 63 Similar observations have been made in humans. Normal serum clearance is -1 ml/min, and the daily excretion of carnitine is 100-300 pmol. Excretion increases after oral administration, and after 6 h -10% of a l-g oral dose is recovered in the urine. Tubular reabsorption in the kidneys was found to be 96-99% (140, 162, 257). Carnitine is also excreted in milk. The concentration in cow’s milk is 0.1-0.5 mM (146), in goat’s milk is -0.1 mM, in sheep’s milk is nearly 1 mM (378), and in human milk is ~0.05 mM (53). No studies on possible losses of carnitine to the gastrointestinal tract have appeared. in Microurganisms Several mechanisms are known for the degradation of carnitine (Fig. can metabolize carnitine by the following 3). Different strains of Pseudomonas reaction (246, 362) (-)carnitine + NAD g y-trimethylamino-P-ketobutyric acid + NADH where NADH is the reduced form of NAD. The enzyme (EC 1.1.1.108) is inducible and specific for (-)carnitine. The product can decarboxylate spontaneously to trimethylaminoacetone. In the presence of ATP and CoA, however, extracts of Pseudomonas convert i(CH331 I CHOH \r I i(CH313 + CH2 I COOH COOH I CH2 I CHOH CH I 2 \IV CO I L COOH I COOH (4 orl-1carnit ine Trimethylamine + Malate Dehydro carnitine i(CH331 I II ~ tilCH313 I CH 2 I COOH + CH3 I COOH Betaine +acetate Trimet CH2 I co I CH3 + CO 2 \ V ihCH3) 3 I CH2 I CHOH I CH3 hylaminoacetone (+) Methyl choline FIG. 3. Catabolism of carnitine. I: Carnitine dehydrogenase. Inducible and specific for (-)carnitine and NAD in some microorganisms. [In insects and mammals both (-)- and (+)carnitine may be oxidized by a similar enzyme.] II: This decarboxylation may be spontaneous (nonenzymatic). III: Cleavage in some microorganisms by unknown enzyme(s). IV: CoA- and ATP-dependent cleavage in some microorganisms. CoA ester(s) are probable intermediate(s). V: Trimethylaminoacetone is reduced to methylcholine in some insects and possibly in mammals. Enzyme unknown. - Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 B. Degradation October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1433 the y-trimethyl-P-ketobutyric acid to glycine and acetate, presumably with the CoA ester(s) as intermediate(s) (246). This reaction sequence can explain Pedicoccus why carnitine can substitute for betaine in the betaine-requiring sogae (358). The microorganism Serratia murcescens can form trimethylamine and malate from carnitine (411). Escherichia coli can reduce carnitine to r-butyrobetaine, presumably by hydrogenation after dehydration of carnitine to y-crotonbetaine (365). in Insects The larvae of some choline -requiring i nsects [Mwca donwstica (housefly), regina (blowfly), and Drosophila melanogaster (fruit fly)] can form methylcholine from carnitine and can use this product instead of choline for phospholipid synthesis (29, 79, 164). Both (-)carnitine and (+)carnitine can be utilized (33, 164), and it is striking that both isomers are converted to (+)methylcholine, showing that the hydroxyl group of carnitine is epimerized in the process (33). To explain this phenomenon, it was suggested that both isomers are converted to trimethylaminoacetone, which subsequently is reduced to (+)methylcholine. The trimethylaminoacetone was not isolated, but it was shown to be a methylcholine precursor. The formation of methylcholine has also been explained by the action of the enzyme carnitine decarboxylase (EC 4.1.1.42), which has been found of this both in rat and in Phormia regina (175, 227). The stereospecificity enzyme is not known because the assays were done with labeled racemic (_+)carnitine. The (+)methylcholine found in the phospholipids, however, would have to be formed from the unphysiological (+)isomer. To complicate matters further, some evidence has been presented for the formation of phosphatidylcarnitine in Phormia regina, and some researchers have suggested that phosphatidylmethylcholine is formed by decarboxylation of phosphatidylcarnitine (275, 277). Phormia D. Degradation in Mammals Feeding experiments in which mice and rats were given heavy oral loads of carnitine have shown increased urinary excretion of trimethylamine and trimethylamineoxide, and even of butyrobetaine and crotonobetaine (367, 392, 393). These metabolites are probably formed by intestinal microorganisms (335, 367). Small amounts of trimethylaminoacetone are found in normal urine (171) and may also be of intestinal, microbiological origin. One striking bit of evidence, however, is that the administration of the unphysiological isomer (+)carnitine to rats and mice leads to a far higher excretion of trimethylaminoacetone than does administration of (-)carnitine, and parenteral admin- Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 C. Degradation 1434 JON BREMER Vohme 63 VII. REGULATION OF CARNITINE TURNOVER Our knowledge of possible regulatory mechanisms in the turnover of carnitine is scarce. In the neonatal period rat pups apparently receive most of their carnitine from mother’s milk (346), and in human babies carnitine probably is an essential nutrition factor (53, 307, 373). In the growing rat, however, the capacity for carnitine synthesis is sufficient, even with no carnitine in the diet (52, 150). It has been shown that a deficiency of lysine or protein leads to a moderately reduced carnitine content in heart, skeletal muscle, and epididymis (54, 228, 396, 400) and that carnitine can promote growth in rats on a low-methionine diet, suggesting that carnitine may have a methionine-sparing action (226). But carnitine synthesis depends on methylated lysine in proteins (235), and only a minor fraction of the methyl groups of methionine is finally incorporated into carnitine (59). It seems unlikely Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 istration is more efficient than administration per OS (366). Thus by some unknown mechanism animal tissues seem to metabolize (+)carnitine to trimethylaminoacetone far more efficiently than (-)carnitine. We have recently confirmed this preference after injection of rats with rH]CH,(+)and (-)carnitine (Fig. 2). We have also found small amounts of a compound that is chromatographed like methylcholine. Previous studies on the metabolism of labeled racemic (+)carnitine showed conversion of carnitine to what was identified as methylcholine (227, 276). The results of Seim et al. (366), which have been confirmed in this laboratory (Fig. l), suggest that these earlier studies really represent primarily the conversion of (+)carnitine to trimethylaminoacetone. However, the role of carnitine decarboxylase partially purified from mitochondria remains unexplained (227). Carnitine decarboxylation reportedly is stimulated in diabetic and fasting rats, and in accordance with this finding the diabetic rats were found to have a decreased body pool and a decreased half-life of carnitine (276). As mentioned above, however, these studies were performed with labeled racemic (&)carnitine. In a more recent study the decreased carnitine content in the skeletal muscles of diabetic rats could not be confirmed (388). There is also other evidence for the degradation of (-)carnitine in mammals. Carboxyl-labeled butyrobetaine, which is rapidly converted to (-)carnitine in the body, gave rise to some radioactive CO2 in rats (60), and [‘4C]CH3(-)carnitine also led to the formation of radioactive COz (247). However, the slow rates of formation of labeled COz make it difficult to exclude participation of intestinal microorganisms. Altogether it is possible that some degradation of (-)carnitine takes place in mammals, but it seems doubtful that this degradation is of any quantitative importance. Most of the carnitine seems to be excreted unchanged in the urine. October 1983 CARNITINE-METABOLISM AND 1435 FUNCTIONS VIII. FUNCTION OF CARNITINE IN MITOCHONDRIAL FATTY ACID OXIDATION In his 1955 paper Fritz (155) identified carnitine as the active component in muscle extracts that had previously been observed to stimulate fatty acid oxidation in perfused livers (255). In subsequent papers carnitine was also shown to stimulate fatty acid oxidation in skeletal and heart particulate fractions, and the effect was shown to be specific for carnitine. A stimulating effect of CoA in the presence of carnitine was also observed (156). By that time it was well known that isolated mitochondria contain the fatty acid @-oxidation enzymes and that mitochondria normally retain CoA and other cofactors through isolation and washing procedures. Because of these different observations and because of the established acetylation of carnitine (153), acetylcarnitine and fatty acid esters of carnitine (61, 62) were tested and were found to be excellent oxidizable substrates for isolated mitochondria. The reversibility of the acetyl group transfer to the mitochondria was demonstrated by the formation of acetylcarnitine in the presence of carnitine and pyruvate (61). It was suggested that carnitine transports activated fatty acids through the mitochondrial membrane and that this carrier function represents an important “loophole” in the compartmentation of the cell (Fig. 4). Subsequently a CoA-dependent formation of palmitoylcarnitine (63) and Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 therefore that protein methylation, which may have other functions, is influenced by the availability of carnitine. After breakdown of the proteins the trimethyllysine is either converted to carnitine (139,235) or excreted in the urine (220). Thus the conversion of trimethyllysine may be regulated, but it is not known whether this conversion is influenced by carnitine. Holme and co-workers (195) have recently reported that the y-butyrobetaine,Z-oxoglutarate dioxygenase is “uncoupled” by carnitine. A similar mechanism might regulate trimethyllysine, Z-oxoglutarate dioxygenase (EC 1.14.11.-), which has a similar reaction mechanism and which represents the initial step in the conversion of trimethyllysine to carnitine (203). In normal animals the excretion in urine of unchanged carnitine seems to be the main pathway of loss. This excretion is increased in thyrotoxic and decreased in hypothyroid patients (256). Possibly this just corresponds to an increased turnover of protein in the hyperthyroid state. At present it seems unlikely that carnitine breakdown is quantitatively important in carnitine turnover in animals, although intestinal microorganisms may degrade this compound. Because plasma carnitine is elevated in kidney failure (44, 108), the kidneys must take part in regulation of the plasma carnitine level. As, mentioned in section VIA, carnitine per OS increases urinary carnitine excretion, whereas fasting first decreases and then increases carnitine excretion. 1436 JON Ceil BREMER membrane i’iium 63 Mitochondrion /3-OH-but t AcAc--- p--c Peroxisome I -AC-CoA FFA h8' -Acyl-CoA + c24 CoA p-ox. = Acyl-CoA C20 G1yc.P FFA c4-c Triacylglyc. P-lip. 1 10 I I FIG. 4. Compartmentation of fatty acid metabolism in liver. Activation of free long-chain fatty acids to acyl-CoA takes place in endoplasmic reticulum or in outer mitochondrial membrane. Circles across mitochondrial membranes represent carnitine-dependent transfer of acetyl and in detail in long-ch ain acyl groups from extramitochondrial to intramitochondrial CoA (shown Fig. 5). In this scheme it is assumed that acetyl-CoA and shortened fatty acids formed in peroxisomes are transported into mitochondria via this carnitine-dependent mechanism. FFA, free fatty acids; Glyc.P, glycerol-3-phosphate; P-lip., phospholipids; Triacylglyc., triacylglycerol; CoA, coenzyme A; Ac-CoA, acetyl-CoA; AcAc, acetoacetate; P-OH-but, p-hydroxybutyrate; pox., B-oxidation. a carnitine-dependent oxidation of palmitoyl-CoA in isolated mitochondria, with palmitoylcarnitine as an intermediate (160), were shown. Even though the carnitine acyltransferases were assumed to be present inside and outside the mitochondria, the carnitine palmitoyltransferase was later shown to be localized exclusively in the inner mitochondrial membrane (305, 306). Functional studies with isolated mitochondria showed that mitochondria contain an external (overt) and an internal (latent) carnitine palmitoyltransferase (3’71, 438), and digitonin extraction showed that the external transferase is more easily solubilized. After such solubilization of the outer transferase the mitochondria can no longer oxidize palmitoyl-CoA in the presence of carnitine, whereas the oxidation of palmitoylcarnitine is unhampered (199). Early studies of mitochondrial permeability suggested that the inner mitochondrial membrane was impermeable to carnitine (65, 176). It was therefore assumed that the inner carnitine palmitoyltransferase is a vecextramitochondrial (acyl-) carnitine to react with torial enzyme permitting intramitochondrial CoA. The isolated heart mitochondria, however, retain Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 FFA clO‘ ------co2 '-Ox- October 1983 CARNITINE-METABOLISM AND 1437 FUNCTIONS Outer membrane Inner membrane Matrix AMP+ PP; RCOO-+ ATP tine FIG. 5. Carnitine-dependent transport of activated fatty acids across inner mitochondrial membrane. I: Acyl-CoA synthase; II: outer carnitine acyltransferase; III: inner (latent) carnitine acyltransferases; and IV: carnitine/acylcarnitine translocase. AMP, adenosine monophosphate; ATP, adenosine triphosphate; CoA, coenzyme A; PPi, inorganic pyrophosphate; RCOO-, longchain fatty acid. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 an intramitochondrial carnitine pool that is exchanged against added extramitochondrial carnitine or acylcarnitines (322,337). Thus the inner membrane of heart mitochondria contains a carnitine translocase catalyzing a one-to-one exchange of carnitine and/or acylcarnitine across the membrane. Later the translocase was also demonstrated in liver mitochondria (329) and in spermatozoa (84). In isolated liver mitochondria the carnitine pool is small, thus preventing detection of carnitine permeation when carnitine space is measured. At present the carnitine-dependent acyl group-transfer system of mitochondria is believed to consist of an outer and an inner carnitine acyltransferase connected by a carnitine translocase (322,337). So far, however, no separate translocase protein has been identified (Fig. 5). It has been suggested that the uptake of acylcarnitine in mitochondria is an energy-dependent process prevented by uncouplers of oxidative phosphorylation (130, 240). However, the observed inhibition of acylcarnitine oxidation in uncoupled mitochondria was probably due to osmotic dehydration of the mitochondrial matrix under the conditions used (316). The function of carnitine in the oxidation of fatty acids must be understood mainly in relation to the cellular localization of the acyl-CoA synthases. The long-chain acyl-CoA synthase was originally found only in the endoplasmic reticulum (234) but was later discovered in the outer membrane of the mitochondria as well (1, 163, 306, 4X), that is, was found outside the permeation barrier to CoA in the inner membrane of the mitochondria. In contrast the short- and medium-chain-length acyl-CoA synthases are localized in the mitochondrial matrix (1, 2, 360). This explains why the oxi- 1438 JON BREMER Vohme 63 Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 dation of short- and medium-chain-length fatty acids is carnitine independent (423). A certain ability of the medium-chain-length acyl-CoA synthase inside the acyl-CoA barrier to activate high concentrations of long-chain fatty acids (371) explains the repeated observation that isolated mitochondria can oxidize long-chain fatty acids in the absence of carnitine (170). This oxidation of long-chain fatty acids, however, is of doubtful physiological significance in the intact cell. The intramitochondrial capacity for activation of longchain fatty acids amounts to only 1% of the extramitochondrial capacity in rat liver (170). The overlapping and organ variation of specificities explain why the oxidation of octanoate is stimulated by carnitine skeletal muscle mitochondria (169). The mitochondria also contain a guanosine 5’-triphosphate (GTP)-dependent acyl-CoA synthase [guanosine 5’0diphosphate (GDP)-forming] in their matrix (348), which theoretically should permit carnitine-independent oxidation of long-chain fatty acids. But careful studies with isolated liver mitochondria have made it doubtful that this enzyme of low activity is of any normal physiological significance in the intact cell (15). Isolated mitochondria can also oxidize long-chain acyl-CoA esters in the absence of carnitine and in the presence of free fatty acids, or when relatively high concentrations of acyl-CoA are used (73, 439); such oxidation presumably can occur because of detergent effects that make the mitochondrial membrane permeable. This carnitine-independent oxidation is probably of no physiological significance. Nevertheless, even if the carnitine dependence of the oxidation of longchain fatty acids in mitochondria is the rule in animals, it is not without exception. Flight muscle mitochondria from a moth (Prodenic eridania) oxidize palmitate at a rapid rate in the absence of carnitine. These mitochondria contain no carnitine palmitoyltransferase but have a very active longchain acyl-CoA synthase (390). Isolated mitochondria from animal tissues normally oxidize acylcarnitines of all chain lengths from C2 to C= (62,317), and they contain carnitine acyltransferases that may show even higher activities with short- and medium-chain-length fatty acids than with long-chain fatty acids (121, 381). Because the medium- and short-chain acyl-CoA synthases are localized in the mitochondria, it has been difficult to understand the function of mediumand short-chain carnitine acyltransferase activities. Again our understanding derives from the overlapping chain-length specificities of the extra- and intramitochondrial acyl-CoA synthases. This overlapping assures that fatty acids of any chain length (including unusual fatty acids with branched chains or other side groups) can be metabolized. The medium- and short-chain carnitine acyltransferase activities ensure that any medium-chain fatty acid CoA ester formed “by accident” outside the mitochondria can be transferred to the mitochondria for oxidation. Note that octanoate oxidation has been found to be carnitine dependent in skeletal muscle mitochondria (169). October IX. 1983 FUNCTION CARNITINE-METABOLISM OF CARNITINE IN ACETATE AND FUNCTIONS 1439 METABOLISM A. Fatty Acid Synthesis B. Acetate and AcetyGCoA Oxidation There is no general agreement on whether carnitine is active in the oxidation of extramitochondrial acetyl-CoA. No significant oxidation of added acetyl-CoA in the presence of carnitine was obtained in isolated mitochondria of ox liver (141) or in the flight muscle of Phomzia regina (109) despite a high carnitine acetyltransferase content. Apparently almost all the enzyme was latent (inner transferase), which permits reaction only with mitochondrial acetyl-CoA. From these and similar results it has been suggested that carnitine acts mainly as a buffer or as a sink for mitochondrial acetyl-CoA, allowing a shift of the “acetyl pressure” from the mitochondria to the cytosol. This has been suggested for tissues as different as sheep liver (375,377), rat heart (333), frog muscle (4), and blowfly flight muscle (109). Because carnitine is usually present in a much higher concentration than CoA, the extramitochondrial acetylcarnitine/carnitine ratio will prevent great fluctuations in the mitochondrial acetyl-CoA/CoA ratio. A similar function for acetylcarnitine as “spare fuel” can be visualized for spermatozoa (see sect. xx). Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The metabolism of acetate and of acetyl-CoA requires specific consideration, because acetyl-CoA synthase is found both-intraand extramitochondrially, especially in lipogenic tissues such as liver, gut, and mammary gland (13, 360), and because acetyl groups are transferred from the mitochondria to the cytosol during lipogenesis. Because acetylcarnitine is formed from carnitine and pyruvate in isolated mitochondria (61, 109, 208), it was assumed that carnitine might be active in the transfer of acetyl groups from the mitochondria to the cytosol for fatty acid synthesis (61). Although the idea gained some support from studies on the effect of carnitine on fatty acid synthesis in guinea pigs (77), this seems generally not to be the case. Even in the guinea pig the activity of the outer carnitine acetyltransferase OF he tota2 (II). In rats and other animals is very low- only a Pew percent the acetyl group transfer from the mitochondria takes place via citrate synthesis in the mitochondria and extramitochondrial citrate cleavage to oxaloacetate and acetyl-CoA by ATP citrate (vo-3S)-lyase (EC 4.1.3.8) (420). In ruminants, which lack citrate lyase, fatty acid synthesis seems to take place from free acetate formed in the rumen (23,182). In addition the highest activities of carnitine acetyltransferase are found in nonlipogenic tissues such as heart, skeletal muscle, and testis, whereas the enzyme’s activity is low in lipogenic tissues such as liver and adipose tissue (264). 1440 JON Vohme BREMER In some studies with isolated mitochondria, however, a rapid oxidation of added acetyl-CoA; this may occur, tochondria from rat heart (161), from pig kidney (58), muscle of the locust (Locusta migratoria) (19). Carnitine be involved in the oxidation of excess extramitochondrial cially acetyl-CoA formed in the peroxisomes. X. FUNCTION OF CARNITINE IN PEROXISOMAL FATTY ACID 63 carnitine stimulates for example, in miand from the flight therefore may also acetyl-CoA, espe- OXIDATION Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The recent demonstration of a P-oxidation system for fatty acids in the peroxisomes (237) suggests a function for the short- and medium-chain carnitine acyltransferase(s), that is, the transfer of acyl-CoA esters formed outside the mitochondria for oxidation in the mitochondria. Isolated peroxisomes have been found to contain carnitine acetyltransferase and medium-chain carnitine acyltransferase but no carnitine palmitoyltransferase (31, 219, 261, 263). Peroxisomal fatty acid oxidation does not require the participation of carnitine. Apparently the peroxisomal membrane contains a direct carrier or a permease for acyl-CoA (5, 319). But peroxisomal fatty acid oxidation is only partial; that is, the fatty acids are shortened (237, 320). This shortening seems to be particularly important in the metabolism of very-longchain fatty acids (e.g., C,), which are poorly oxidized in mitochondria (74, 118, 119). The reaction products (acetyl-CoA and a shortened acyl-CoA) must be transferred to the mitochondria for complete oxidation. It is therefore reasonable to assume these products are transferred via the carnitine acyltransferases. Because carnitine acyltransferases are present in peroxisomes, a transfer of this acetyl-CoA and of the shortened acyl-CoA would not require the presence of external transferase(s) in the mitochondria. A direct demonstration of such transfer is still lacking, however, although some evidence has been obtained for very-long-chain fatty acids. It has been shown that the chain shortening of erucic acid (C,) is unaffected by (+)decanoylcarnitine, an inhibitor of carnitine-dependent fatty acid oxidation in the mitochondria, or by carnitine depletion of the cell, but that the complete oxidation of erucic acid to ketone bodies and CO2 is carnitine dependent (118). In this connection it is striking that clofibrate, which induces increased peroxisomal oxidation of fatty acids in the peroxisomes, also induces increased activity of the carnitine acyltransferases (particularly the acetyltransferase) in both mitochondria and peroxisomes (219,262,289,382). AcylCoA hydrolases in the cytosol are also induced under these conditions (49). Therefore the transfer of the peroxisomal products to the mitochondria as free acetate and free fatty acids is also a possibility. October XI. HOW 1983 MANY CARNITINE-METABOLISM CARNITINE AND FUNCTIONS 1441 ACYLTRANSFERASES? Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The varying ability of mitochondria from different tissues to oxidize acylcarnitines of different chain lengths suggested that more than one carnitine acyltransferase existed (62). This assumption was first confirmed by the separation of carnitine acetyltransferase from carnitine palmityltransferase (159, 160, 297, 298). The separated enzymes have overlapping chainlength specificities. Shortly afterward the carnitine acetyltransferase was prepared in crystalline form (103). The palmitoyltransferase proved to be more difficult to purify, but homogeneous preparations of this enzyme have also been prepared (121, 233). After the initial studies, a bewildering number of different carnitine acyltransferases were postulated, first in mitochondria and later also in peroxisomes and in other parts of the cell. The demonstration by Yates and Garland (438) of different functional pools of the transferases [one easily soluble outer transferase and one firmly bound inner transferase (199)] in the mitochondria led to attempts to characterize these different pools. West et al. (421) found that the outer palmityltransferase was inhibited by Zbromopalmitoyl-CoA, whereas the inner transferase was not. This difference in sensitivity still remains unexplained. Fritz et al. (83, 233) found immunological and kinetic differences between the isolated inner and outer transferases and suggested that the outer enzyme could be converted to the inner enzyme. Studies of chain-length specificity that were performed on intact mitochondria and on soluble enzyme preparations have shown essentially three activity peaks depending on fatty acid chain length. One peak occurs at chain lengths C2-3 (acetyltransferase), one at C &l() [octanoyl transferase (EC (232, 380-382). Thus these 2.3.1.-)], and one at C 14-16 (palmitoyltransferase) studies imply a total of six carnitine acyltransferases in mitochondria (i.e., 3 outer and 3 inner enzymes). In addition carnitine acetyltransferase and medium-chain carnitine acyltransferase have been found in peroxisomes (31, 224, 262, 263, 344) and in the endoplasmic reticulum (261), and palmityltransferase has been reported to be present in the cell membrane of erythrocytes (428), cells that lack intracellular organelles. Recent studies, however, again seem to reduce the number of carnitine acyltransferases. In a careful study Bergstrom and Reitz (24) have shown that outer and inner palmitoyltransferases purified with identical chromatographic procedures most likely are identical proteins. Clarke and Bieber (121) also used chromatographic procedures to show that heart mitochondria contain only two carnitine acyltransferases, one short-chain acyltransferase and one long-chain acyltransferase, again with overlapping chain-length specificities. They have also shown that in heart the octanoyl transferase activity may be explained by this overlap and by the kinetic properties of the long-chain transferase. Recently, however, a separate octanoyl transferase 1442 JON BREMER Vobmne 63 XII. CARNITINE A. Isolation ACETYLTRANSFERASE and Properties Carnitine acetyltransferase is relatively easily extracted from the mitochondrial membrane with salt solutions, and it has been crystallized from pigeon breast muscle (103). The molecular weight of the enzyme from pigeon breast muscle is -55,000, from beef heart is 62,000 (121), and from rat liver with slightly is 56,000 (282). In Candidu tropicalis two acetyltransferases different properties have been isolated, one from mitochondria and the other from peroxisomes. Both enzymes have two subunits with molecular weights of -60,000 and 70,000 (410). The rat liver enzyme also appears to have two subunits with molecular weights of 34,000 and 25,000 (282). Only one of the subunits has a catalytic site; the function of the second subunit is not known. B. Kinetics and SpeciJicity The reaction acetyl-CoA + carnitine e acetylcarnitine + CoA is freely reversible, with the equilibrium constant (K,,-J equal to 0.6-0.7 (68, 159). The K,, decreases with the chain length of the acyl group. The Keg value close to unity shows that the free energy of the O-ester bond of acetylcarnitine is similar to that of the thioester bond of acetyl-CoA, or cit. -7.9 kcal/mol. This unusually active character of an O-ester bond is explained by the inductive effect caused by the closeness of the molecule’s quaternary Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 has been purified to homogeneity from mouse liver peroxisomes (148). This third transferase probably is also present in liver mitochondria (381). Edwards and co-workers (141) have presented evidence indicating that mitochondria contain only one acetyltransferase. Thus the number of carnitine acyltransferases in heart mitochondria at present seems to be back to the original two, whereas in liver three transferases have been isolated. It is still uncertain whether the extramitochondrial transferases in animal tissues represent unique enzymes. Nevertheless Japanese workers have and peroxirecently shown that Candida tropic&& contains mitochondrial somal carnitine acetyltransferase; in their purified forms these enzymes have different molecular weights and can be separated with chromatographic and electrophoretic procedures (224, 410). Further work is needed to establish how the extramitochondrial transferases are related to the two firmly established mitochondrial ones. October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1443 C. Inhibitors and “Suicidal” Substrates Bromoacetylcarnitine and bromoacetyl-CoA inhibit the enzyme rapidly, and studies of their effects have shed light on the reaction mechanism of Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 nitrogen (148a). Chase and Tubbs (101,102,104-106) have thoroughly studied the kinetics and reaction mechanism of carnitine acetyltransferase. The following K, values for the four substrates (2 in each direction) have been found: carnitine, 120 PM; acetyl-CoA, 37 PM; acetylcarnitine, 350 PM; and CoA, 37 PM. The reaction products in one direction are competitive inhibitors of the corresponding substrates in the same direction. The enzyme has a relatively broad pH optimum between 7 and 8.5. The enzyme shows a pronounced specificity for CoA and carnitine, but it does react with norcarnitine (P-hydroxy-y-dimethylaminobutyric acid) (158) or with dephospho-CoA (lo@, although these compounds have much higher K, values than its physiological substrates. Thiocarnitine (P-sulfhydryl-ytrimethylaminobutyric acid) also reacts with the enzyme (149). The K, values for the different straight-chain acyl-CoA substrates are nearly independent of acyl length. The enzyme from pigeon breast muscle shows the highest reaction rate with acetyl-CoA and with gradually declining rates as the chain length of the acyl gro.up increases. With decanoyl-CoA the rate is only 4% of that with acetyl-CoA. The rat heart and pig heart enzymes show the highest rates with propionyl-CoA (121). The enzymes from Candida tropicalis react almost exclusively with acetyl-CoA. The rate with propionyl-CoA is only a few percent of that with acetyl-CoA (410). Thus the Candida enzymes have a much narrower chain-length specificity than the animal enzymes. The enzyme from animal tissues also reacts with branched-chain acyl groups, because branched-chain acylcarnitines are formed when mitochondria are incubated with branched-chain cu-keto acids (383). Acetoacetylcarnitine is also a substrate for the enzyme. Because of the low concentration of acetoacetyl-CoA in the tissues, however, it is unlikely that acetoacetylcarnitine is formed, although it has been used as a substrate in experimental studies of ketogenesis (68). Long-chain acyl-CoA esters inhibit the enzyme (102). This inhibition has a peculiar feature: palmitoyl-CoA acts as a competitive inhibitor toward both carnitine and acetyl-CoA. Free palmitate is also a weak competitive inhibitor (but surprisingly only against carnitine). These results have been interpreted to mean that the enzyme has a hydrophobic site that binds long-chain acyl groups and that enhances the binding of the CoA moiety. At the same time the long-chain acyl group prevents the binding of carnitine to its reactive is as site. In accordance with this interpretation the Ki of palmitoyl-CoA low as 1 PM, whereas for palmitate it is several hundred times higher. 1444 JON BREMER vohmie 63 XXII. CARNITINE OCTANOYLTRANSFERASE Carnitine octanoyltransferase #Zen.eity from mouse liver peroxisomes, (EC 2.3.1.-) has been purified to homofrom which it can be extracted without Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 the enzyme. Bromoacetylcarnitine can inactivate the enzyme by two mechanisms. In the presence of CoA its -SH group is alkylated on the enzyme active site, and an S-carboxymethyl-CoA carnitine ester is formed. It is firmly bound to the enzyme [dissociation constant (Kd) = 10-lo], and the enzyme activity is blocked. With bromoacetylcarnitine alone (in the absence of CoA) a histidine residue in the enzyme is irreversibly alkylated. These results confirm that the enzyme forms a ternary complex with its substrates and that a histidine residue takes part in its catalytic process at the carnitine site (105, 106). With bromoacetyl-CoA (in the presence of carnitine) bromoacetylcarnitine is first formed by the enzyme. The -SH group of CoA is then alkylated, as with added bromoacetylcarnitine. Thus bromoacetyl-CoA is a “suicidal” substrate for the enzyme (105). (+)Carnitine and (+)acetylcarnitine are competitive inhibitors of the enzyme when tested against (-)carnitine and (-)acetylcarnitine. [The Ki values are 170 and 250 PM, respectively (104)]. Fluoroacetylcarnitine is as good a substrate for the enzyme as acetylcarnitine. In isolated mitochondria fluoroacetyl-CoA is rapidly formed in the matrix; intramitochondrial fluorocitrate is presumably formed, and the citric acid cycle is inhibited. Fluoroacetylcarnitine therefore can be used to inhibit the citric acid cycle in isolated heart mitochondria, for example, where added fluorocitrate has no effect because the mitochondrial membrane is impermeable to fluorocitrate (69). Pent-4-enoylcarnitine reacts rapidly with carnitine acetyltransferase and is converted to pent-4-enoyl-CoA in the mitochondria, where it is further metabolized to pent-2,4-dienoyl-CoA and 3-keto-pent-4-enyl-CoA. Both these compounds are inhibitors of acetyl-CoA acyltransferase (EC 2.3.1.16) and therefore of fatty acid oxidation (194,363). Low concentrations of pentenoate do not inhibit fatty acid oxidation because the compound can be completely metabolized by a separate route. This metabolism of pentenoate depends on a reduction of the pent-2,4-dienoyl-CoA to pent-Z-enoyl-CoA with reduced NAD phosphate (NADPH) by 2,4-dienoyl-CoA reductase, which is followed by a normal P-oxidation (50,190). Here NADPH can be supplied from NADH via the energy-dependent transhydrogenase in mitochondria. This is analogous to the function of this enzyme in the metabolism of unsaturated fatty acids (318). These findings explain why clofibrate treatment, which dramatically increases the activity of 2,4-dienoyl-CoA reductase (EC 1.3.1.-), protects against toxic effects of pent-4-enoylcarnitine and why uncoupling increases the inhibitory effect of pent-4-enoylcarnitine in isolated mitochondria (50). October CARNITINE-METABOLISM 1983 AND FUNCTIONS 1445 XIV. CARNITINE PALMITOYLTRANSFERASE A. Purification and Pvwpmties Carnitine palmitoyltransferase is more firmly bound to the mitochondrial membrane than is carnitine acetyltransferase, but its properties are in many ways similar. Norum (298) obtained a partially purified enzyme by salt extraction of lyophilized, washed calf liver mitochondria, but highly purified preparations of the enzyme are obtained only by the use of detergents (24, 121, 232, 233). Kopec and Fritz (233) isolated the enzyme from calf liver in two forms, one of which was very labile and was probably a partially denatured enzyme. The enzyme was found to be a dimer with a molecular weight of 2 X 75,000. Bergstrom and Reitz (24) and Clarke and Bieber (121) isolated the enzyme from rat liver and beef heart, respectively, as polymers with molecular weights of 400,000-500,000 and with subunit weights of ~65,000. The amino acid compositions of the acetyl- and palmitoyltransferases were found to be similar (121). It is not yet known whether all subunits have catalytic sites. B. Kinetics and Spea$icity Like the acetyltransferase, the palmitoyltransferase reacts with norcarnitine (300) and with dephospho-CoA (76) but with reduced reaction rates. A peculiar feature of the palmitoyltransferase is that one of its substrates (palmitoyl-CoA) behaves as a competitive inhibitor to its cosubstrate Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 the use of detergents (148). Its molecular weight is ^160,000. It is stable in pyrophosphate buffer, but it is rapidly inactivated by freezing. Its chain-length specificity overlaps those of the acetyl- and palmitoyltransferases. Thus the maximum reaction rates with acetyl-CoA and palmitoyl-CoA are 26 and 8%, respectively, of that with octanoyl-CoA. The relative reaction rates in the reversed direction are similar. The & value for octanoyl-CoA is 15 PM (for acetyl-CoA and palmitoylCoA the Km values are 130 and 70 PM, respectively) and for carnitine is 155 and CoA PM. In the reverse direction the Km values for octanoylcarnitine are 100 and 780 PM, respectively. The activity of octanoyltransferase in the liver is increased by clofibrate and other hypolipidemic drugs, as are the activities of the other carnitine acyltransferases (148). The effects of inhibitors on the purified enzyme have not been studied, but in isolated mitochondria malonyl-CoA inhibits the formation of octanoylcarnitine from octanoyl-CoA (254, 353). 1446 JON BREMER Vohme 63 carnitine, thus increasing the apparent K, of carnitine (71,72). This property of the enzyme, which is also present in intact mitochondria (73,436), explains a strong substrate inhibition by palmitoyl-CoA in the forward reaction (the reaction of the outer carnitine palmitoyltransferase) palmitoyl-CoA + carnitine - palmitoylcarnitine and a strong product inhibition of the reversed reaction inner palmitoyltransferase) ’ + CoA - palmitoyl-CoA (the reaction of the + carnitine The possible physiological significance of this enzyme property is that it may act to slow down the transfer of acyl groups to the mitochondrial matrix when a rapid flux of fatty acids into the cell gives a high extramitochondrial level of acyl-CoA. Thus “flooding” of the mitochondria with longchain acyl-CoA may be prevented. The inhibitory effect of palmitoyl-CoA on carnitine binding to the enzyme is evidently similar to the effect of palmitoyl-CoA on carnitine binding the difference is that palmito carnitine acetyltransferase (see sect. XII@; toyl-CoA is also the substrate of the enzyme in the case of palmitoyltransferase. Hence palmitoyltransferase, like acetyltransferase, seems to contain a hydrophobic region binding the long-chain acyl groups of the substrates. This binding evidently contributes to the substrate binding and explains why the K, values for acylcarnitines and acyl-CoA esters decrease with the length of the fatty acid (102, 232). With palmitoyl-CoA the following K, values have been found for the four substrates: palmitoyl-CoA, lo-20 PM; carnitine, 250-450 PM (extrapolated to 0 palmitoyl-CoA); palmitoylcarnitine, 40-140 PM; and CoA, 5-50 (71, 232). PM (5 PM when extrapolated to 0 palmitoylcarnitine) The spread of the determined values probably has to do with the low solubility and the micellar properties of the long-chain acyl substrates and the complicated kinetics of the enzyme. The acyl group chain-length specificity of carnitine palmitoyltransferase is apparently different in the two directions of the reversible reaction (122). In the forward reaction (acylcarnitine formation) the optimum chain length of the acyl-CoA was Co, whereas in the reverse reaction palmitoylcarnitine gave the highest rate (122, 381). Increasing substrate inhibition, which was due to a lowered Ki of acyl-CoA as the length of the acyl group is increased, most likely explains this phenomenon. The enzyme reacts with a variety of acyl derivatives, although often with reduced rates. Mono-CoA esters of long-chain dicarboxylic acids react with carnitine palmitoyltransferase, but carnitine esters of long-chain dicarboxylic acids are poor substrates for mitochondria (334). Recent results indicate that long-chain dicarboxylic acids are preoxidized (shortened) in Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 palmitoylcarnitine + CoA October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1447 the peroxisomes. Their oxidation is therefore carnitine independent (290). @Keto- and @hydroxyacyl-CoA (75, ZOO,258) are substrates for the enzyme. Carnitine has also been shown to increase the breakdown of prostaglandins (217) and cyclopropane carboxylic acids (173) in isolated mitochondria. A bulky acyl group like pyrenebutyrylcarnitine, however, is not a substrate for the enzyme (432). C Eflect of Detergents D. Pmperties in Mitochondrial Membrane The kinetic properties of carnitine palmitoyltransferase in the intact mitochondrial membrane deviate in important ways from the properties of the soluble enzyme. A physiologically important property of the membranebound outer transferase is its inhibition by malonyl-CoA (270). Little or no such inhibition is found with the soluble enzyme. In the presence of malonylCoA the substrate saturation curve with palmitoyl-CoA changes to a sigmoid form (67, 354). The inhibition by malonyl-CoA is more pronounced in heart and skeletal muscle than in liver and kidney (353). Inhibition is prevented by disulfides such as 3,3’-dithiobis[6nitro-lbenzoic acid (DTNB) (354), suggesting that -SH groups in the enzyme or in the membrane are involved. Disulfides can also inhibit the solubilized enzyme, however (301). Solubilization of the enzyme with detergents also abolishes the malonyl-CoA inhibition (351). The activity of the outer carnitine palmitoyltransferase is modified by K+ and Mg+, whereas these ions have no effect on the solubilized enzyme (351). In liver the inhibition by malonyl-CoA also seems to be modified in different nutritional and endocrine states (see sect. XVII.@. The activities of the outer and inner palmitoyltransferases have been measured indirectly in intact mitochondria by observing the reduction rates of the mitochondrial flavoproteins (acyl-CoA dehydrogenases and electrontransfer protein). The outer transferase activity is measured as the flavo- Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 The kinetics of carnitine palmitoyltransferase are strongly influenced by detergents, partly because of interaction with long-chain acyl substrates. Nevertheless direct effects on the enzyme (via its hydrophobic binding site?) are also a possibility (72,122). A peculiar feature of detergent effects on the enzyme is that the inhibitory effect of palmitoyl-CoA on carnitine binding is more affected by detergents than is palmitoyl-CoA’s function as substrate; that is, detergents increase the Ki of palmitoyl-CoA more than its Km. Thus with low palmitoyl-CoA concentrations, detergents inhibit the enzyme; with high concentrations, detergents have a strong activating effect. The strongest activating effect was obtained with (+)palmitoylcarnitine, the unphysiological isomer of one of its substrates (72). 1448 JON BREMER Vohme 63 E. Inhibitors 2-Tetradecyl glycidate is a potent inhibitor of the outer carnitine palmitoyltransferase and hence of ,&oxidation. At a concentration of 1 PM this substance causes 80-90s inactivation of the transferase. The CoA ester of 2tetradecyl glycidic acid is probably the active inhibitor (409). 2Substituted oxiran-2-carbonoyl-CoA esters are potent inhibitors of the outer carnitine palmitoyltransferase (14). At concentrations of 30-300 nM, some of these CoA esters reportedly cause 50% inhibition of palmitoyl-CoA oxidation by isolated liver mitochondria; such data indicate that these compounds are some of the most potent known inhibitors of the outer transferase. The inner transferase is not inhibited, as judged by the absence of inhibition of palmitoylcarnitine oxidation. Palmitoyl-CoA analogues in which the fatty acyl carbonyl group (or CoA thioester moiety) has been replaced by a methylene group are also potent inhibitors of the outer carnitine palmitoyltransferase (120). 2-Bromopalmitoyl-CoA in the presence of carnitine inhibits the outer carnitine palmitoyltransferase in intact mitochondria. This inhibition presumably occurs by a similar mechanism because the acetyltransferase is inhibited by bromoacetyl-CoA. The inner transferase is not inhibited, however, even after partial purification (107, 421). Note that bromopalmitate is not a specific inhibitor of carnitine palmitoyltransferase in the intact cell. In isolated hepatocytes bromopalmitate appears to inhibit the transport of fatty acids across the cell membrane, possibly by competing with normal fatty acids for binding to the fatty acid-binding protein in the cytosol(260). This process is believed to be essential for fatty acid uptake in the cells. Recent evidence suggests that the outer and inner palmitoyltransferases are identical enzymes (24). If this is so, the inner transferase must be protected against bromopalmitoyl-CoA, even after solubilization and partial purification (421). This discrepancy remains’ unexplained. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 protein-reduction rate after addition of palmitoyl-CoA and carnitine, and the inner transferase activity is measured as the reduction rate after addition of palmitoylcarnitine (296). In such studies the activity of the inner transferase has been found to exceed the activity of the outer transferase by a factor of 500-1,000, both in brown adipose tissue mitochondria (296) and in liver mitochondria (24). After extraction and solubilization, however, the ratio of the same activities is only -5:l (24). These different studies show that the activity of the transferase is profoundly influenced by its environment in the membrane. In this connection it should be remembered that the P-oxidation enzymes in the mitochondrial matrix may be organized in an enzyme complex (387). Studies of the activity of the inner carnitine palmitoyltransferase in situ suggest that this enzyme may be an integrated part of the @-oxidation complex. October XV. 1983 CARNITINE A. Properties CARNITINE-METABOLISM AND FUNCTIONS 1449 TRANSLOCASE and Kinetics B. Inhibitors The 16-trimethylaminohexadecanoyl ester of DL-Carnitine inhibits the action carnitine translocase with a Ki value of -1 PM (408). The inhibitory may be due to the inability of the carnitine/acylcarnitine exchange carrier to transport the cationic acyl group through the inner mitochondrial membrane. Sulfobetaines (N-alkyl-NJV-dimethyl-3-ammonio-l-propane-sulfonates) are also potent inhibitors of the carnitine/acylcarnitine exchange carrier (328). Consequently these compounds are potent inhibitors of mitochondrial fatty acid oxidation. Acyl esters of (+)D-carnitine are potent inhibitors of fatty acid oxidation in isolated mitochondria (157), in isolated hepatocytes (115), and in perfused livers (271). The solubilized carnitine palmitoyltransferase is not inhibited to the same extent (IYI). With isolated mitochondria the inhibition is apparently due to inhibition of the carnitine translocase (322). Pyrenebutylcarnitine, which has useful spectroscopic properties, is a Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Carnitine translocase catalyzes a one-to-one exchange of carnitine and acylcarnitines across the inner mitochondrial membrane (323, 324, 338). A specific carnitine translocase protein has not been isolated, but its existence as a separate entity is assumed on the basis of functional studies in intact mitochondria. The translocase apparently also catalyzes a slow unidirectional transport of carnitine (324, 338). The Km for external carnitine is ~0.5-1.5 mM when measured close to O°C and is much lower for acylcarnitines (209). The Km decreases with the chain length of the fatty acid (329). The exchange rate also depends on the intramitochondrial concentration of carnitine. The exchange is 5-10 times faster with the (-)carnitine isomer than with the (+)isomer [in contrast to the active transport of carnitine across the liver cell membrane, which shows little or no stereospecificity (116)]. is in the same range The Km for carnitine exchange in the mitochondria as the concentration of carnitine in the tissues, and the lower Km for acylcarnitines indicates that the acylcarnitines are preferentially transported into the mitochondria. At 37OC the exchange capacity probably exceeds the capacity of the mitochondria to oxidize fatty acids with a wide margin. At low tissue concentrations of acylcarnitines, however, the translocase may be rate limiting for fatty acid oxidation, and the intramitochondrial acylCoA concentration may be kept low by a significant difference in the extraand intramitochondrial acylcarnitine/carnitine ratios. 1450 JON Volume BREMER very potent inhibitor of the i nner carnitine palmitoyltransferase exch .ange (432). of the acylcarnitine/carnitine XVI. ACYLCARNITINE 63 and also HYDROLASE XVII. REGULATION OF FATTY ACID OXIDATION IN LIVER Numerous studies on perfused livers, isolated liver cells, and perfused hearts have shown that the rates of fatty acid oxidation and of ketogenesis depend on the concentration of fatty acids offered to the tissues (114, 271, 314). Increased rates of fatty acid oxidation also correlate with high levels of long-chain acylcarnitines in the tissues (43, 333). The high level of acylcarnitine may be a direct consequence of the increased uptake of fatty acids. Hence lipolysis in adipose tissue is an important regulatory process with respect to rates of fatty acid oxidation in all animal tissues. The rates of fatty acid oxidation and ketogenesis also depend, however, on the nutritional and endocrine state of the tissue, especially in the liver. This is illustrated by the findings that isolated perfused livers or isolated hepatocytes from fasted (271), diabetic (274), or thyrotoxic animals (385) oxidize more and esterify less of the incoming fatty acids than do livers from normal animals fed carbohydrates. Perfused livers from female rats esterify relatively more and oxidize relatively less of the incoming fatty acids than do livers from male rats (379). Feeding the antilipemic drug clofibrate to rats also increases oxidation and decreases esterification of fatty acids (115). Changes in both cellular metabolites and cofactors and in the mitochondrial carnitine-dependent transport system for activated fatty acids seem to explain these shifts in the metabolism of fatty acids. A. Eflect of Metabolites and Cofactors 1. Makmyl-CoA (270); Malonyl-CoA strongly inhibits the outer carnitine palmitoyltransferase it also inhibits carnitine acetyltransferase in liver but not in other Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 An enzyme hydrolyzing the fatty acid esters of (-)carnitine but not the esters of (+)carnitine has been found in the microsomal fraction of liver, kidney, pancreas, small intestine, and brain (21,259). Acylcarnitine hydrolase (EC 3.1.1.28) hydrolyzes carnitine esters with chain lengths of C6-C18, with Cl0 giving the highest rate. The K, for acylcarnitines is 3-5 mM. Thus one would expect the enzyme to have a low activity in intact cells. Although the function of the enzyme is not known, Mahadevan and Sauer (259) suggested that it prevents excessive accumulation of long-chain acylcarnitines in the tissues. October CARNITINE-METABOLISM 1983 AND FUNCTIONS 1451 tissues (254). In intact animals (174, 443) and in isolated hepatocytes (123, 269) there is an inverse relation between malonyl-CoA content and fatty acid oxidation rates. Insulin stimulates acetyl-CoA carboxylase and increases malonyl-CoA concentration, whereas glucagon has the opposite effect (28); therefore a decreased level of malonyl-CoA is probably important in the stimulation of fatty acid oxidation in hepatocytes isolated from fasted or diabetic rats or in hepatocytes stimulated with glucagon (114). Nevertheless several observations discussed below suggest that additional mechanisms modify the effect of malonyl-CoA. It has repeatedly been suggested that glycerophosphate may inhibit fatty acid oxidation by trapping the acyl-CoA for triacylglycerol synthesis, thus preventing the formation of acylcarnitine. This possibility seems likely from studies of how small amounts of glycerol affect fatty acid oxidation in isolated hepatocytes with a previously low glycerophosphate level (133,253). In intact animals, however, no inverse relationship between the glycerophosphate level and fatty acid oxidation has been found (443). Therefore any variable competing or trapping effect of fatty acid esterification must more likely be explained by varying activities of the triacylglycerol-synthesizing enzymes (see sect. XVII@. 3. &enzyme A In liver the total concentration of CoA about doubles both in whole tissue and in the mitochondria during fasting (374). The initial (and ratelimiting) step in the biosynthesis of CoA is pantothenate kinase (EC 2.7.1.33). The enzyme is inhibited by acetyl-CoA (179). This seems paradoxical because the concentration of acetyl-CoA in the liver rises in fasting animals (407). But it is the amount of mitochondrial acetyl-CoA that increases in fasting and ketotic states, whereas the cytosolic acetyl-CoA/CoA ratio probably decreases (369). In this way the activity of pantothenate kinase will increase and more phosphopantothenate, phosphopantetheine, and CoA will be formed. The mitochondrial CoA is synthesized from the extramitochondrial phosphopantetheine transferred to the mitochondria, where synthesis of mitochondrial CoA is completed (372). Owing to the close correlation between the level of long-chain acyl-CoA in tissues and the rate of fatty acid oxidation (51, 369, 407), it seems likely that the availability of CoA is important. 4 Carnitine Fasting or diabetes increases the level of carnitine in the liver (272). This increase is particularly pronounced in sheep. The carnitine concentration Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 2. Glycerophosphate 1452 JON BREMER t%hmne 63 5. Fatty acid-binding protein Perfused livers from female rats take up and esterify relatively more and oxidize fewer fatty acids than do livers from male rats (379). These differences correlate with a higher concentration of a specific fatty acidbinding protein in the livers of female rats. The level of this protein is increased by estradiol and lowered by testosterone (309). Clofibrate also increases the level of this protein (345). In the cytosol of rat liver the concentration of fatty acid-binding protein amounts to 4-5% of the total protein (310). From studies with isolated hepatocytes, Wu-Rideout and co-workers (437) found that flavispidic acid, which competes with free fatty acids for binding to the fatty acid-binding protein, increases fatty acid oxidation while Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 in the livers of diabetic sheep may be more than lo-fold that of normal sheep (377). The regulatory importance of such data is uncertain. Carnitine can stimulate fatty acid oxidation in isolated hepatocytes because these cells lose most of their carnitine during preparation (114). An increase in carnitine seems “unnecessary,” however, because isolated hepatocytes from fasted rats require a lower concentration of carnitine to give maximum rates of fatty acid oxidation than do hepatocytes from rats fed carbohydrate (117). In intact rats the liver carnitine has been increased about fivefold by intravenous injection of carnitine. This increase in liver carnitine led to increased levels of long-chain acylcarnitines, long-chain acyl-CoA, and acetylcarnitine in the liver; no change in ,&hydroxybuty rate was observed, suggesting that the rate of fatty acid ox .idation remained unchanged (57). It was suggested that an increased transfer of activated fatty acids into the mitochondrial matrix could explain the increased level of acyl-CoA. The authors concluded that this transfer could not be rate limiting in fatty acid oxidation. There is a possible interpretation of these results: The extramitochondrial acylcarnitine/carnitine ratio is in equilibrium with the intramitochondrial acylcarnitine/carnitine and acyl-CoA/CoA ratios via the active carnitine translocase and the inner carnitine palmitoyltransferase. The lessactive outer transferase (and malonyl-CoA) determines the extent to which the extramitochondrial acyl-CoA/CoA ratio deviates from equilibrium with the acylcarnitine/carnitine ratio and the acyl-CoA/CoA ratio of the mitochondrial matrix. In such a system the rate of fatty acid oxidation can be visualized as being regulated both by th e activity of the outer tra .nsferase, which influences the level of oxidizable acylcarni tin es, a ‘nd by the competing subst rates, which will influence the redox situation in the electron-transport chain in the mitochondria and the availability of cofactors (oxidized electron-transfer protein, NAD, and CoA); thus the oxidation rate at a certain acylcarnitine level can be determined. October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1453 it inhibits esterification. Fatty acids bound to this protein therefore seem to be more available for activation and esterification in the endoplasmic reticulum than for activation, acylcarnitine formation, and oxidation in the mitochondria. The mechanisms of these effects are not known. 6. Competing oxidizable substrates B. Changes in Mitochmzdria Liver mitochondria isolated from fasted rats show an increased activity of outer carnitine palmitoyltransferase in the inner membrane and a decreased activity of glycerophosphate acyltransferase (EC 2.3.1.15) in the outer Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Lactate is a strong inhibitor of fatty acid oxidation in hepatocytes from fasted rats. Still, lactate has only a marginal effect on the malonyl-CoA level in hepatocytes from fasted rats (273). Octanoylcarnitine is taken up and oxidized by isolated hepatocytes. In this oxidation only the carnitine translocase and the inner carnitine acyltransferase are supposed to be active. Nevertheless the oxidation rate is stimulated by fasting, by glucagon, and by anti-insulin serum to a similar extent, as is the oxidation of oleate (274). In the mitochondria the incoming acyl-CoA must compete with other substrates for a shared pool of NAD and for a shared electron-transport chain. As a result it is feasible that a variable availability of such substrates or a variable activity of competing enzymes will influence the rate of fatty acid oxidation. Such effects have been demonstrated. Activation of pyruvate dehydrogenase (lipoamide) (EC 124.1) with dichloroacetate accelerates glucose oxidation and inhibits fatty acid oxidation in perfused hearts (267). In isolated liver mitochondria the NAD-linked intermediates of the citric acid cycle have only a weak effect, but high concentrations of the flavoproteinlinked succinate can suppress oxidation of palmitoylcarnitine (75). However, the allover competing ability of the citric acid cycle is weak. On the contrary, a rapid fatty acid oxidation suppresses the citric acid cycle (239, 250, 251). Long-chain acylcarnitines are also dominating substrates compared with pyruvate (64) and glutamate (112). The extramitochondrial NADH is a more efficient competing substrate than the NADH generated within the mitochondria (252), a seeming paradox explained by the active transfer of reducing equivalents into the mitochondria by the malate/aspartate shuttle. The active transport of aspartate out of the mitochondria combined with transamination will efficiently transfer an elevated NADH/NAD ratio in the cytosol to the mitochondria. Because lactate and ethanol generate extramitochondrial NADH, this may be a mechanism by which lactate (270) and ethanol (312) suppress fatty acid oxidation. This inhibition by ethanol seems to take place without any decrease in the acylcarnitine/carnitine ratio in the liver (36). 1454 JON BREMER Vohmw 63 Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 membrane. Refeeding of carbohydrate reverses these effects (16,47,48). The outer carnitine palmitoyltransferase also has a higher activity in the livers of male rats than in those of female rats (356). The activity of the mitochondrial glycerophosphate acyltransferase is also decreased in diabetes, and its activity again increases after treatment with insulin (16). Because the glycerophosphate acyltransferase has a lower Km for acyl-CoA than carnitine palmitoyltransferase, a preferential acylation of glycerol phosphate in the presence of both glycerol phosphate and carnitine can be predicted [as demonstrated in isolated mitochondria (47, 48)]. The inhibitory effect of malonyl-CoA on fatty acid oxidation is decreased in liver homogenates from fasted rats (124, 313). This decreased effect of malonyl-CoA is partly due to the increased activity of the outer carnitine palmitoyltransferase and partly due to a decreased sensitivity of the enzyme (Fig. 6; 67, 353). The inner carnitine palmitoyltransferase shows no or little change in its activity after 24 h of fasting. The enzyme therefore shows a changed distribution (less latency) in the inner mitochondrial membrane after fasting. Consequently it is tempting to suggest that more enzyme that is less sensitive to malonyl-CoA is exposed on the membrane surface. Recently we have found similar behavior of carnitine acetyltransferase in rat liver (254). In liver mitochondria from rats fasted for a long period (48 h) or from diabetic rats an increased total (outer + inner) carnitine palmitoyltransferase has been observed. This increase was not prevented by inhibitors of protein synthesis (299). A similar observation has been done in ketotic and nonketotic alloxan-diabetic rats. In this case increased activity of both the outer and inner enzymes was found (185). In streptozocin-diabetic rats, however, no significant increase in the activity of the outer transferase was found, nor was its sensitivity to malonyl-CoA changed (352). In hepatocytes in tissue culture the activity of palmitoyltransferase has been reported to increase after stimulation by glucagon and to decrease after stimulation by insulin (183). Liver mitochondria isolated from rats treated for two days with 3,5,3’triiodothyronine (hyperthyroid rats) show about a doubling of the activity of the outer carnitine palmitoyltransferase. Mitochondria from thyroidectomized rats or from rats treated with 6-propyl-2-thiouracil (hypothyroid rats) show a decreased activity. Total transferase showed no or little change, and the malonyl-CoA sensitivity was also essentially preserved independent of the thyroid state (357, 385, 386). In agreement with these changes, the mitochondria from the hyperthyroid rats oxidize palmitoyl-CoA in the presence of carnitine 3-4 times faster than do mitochondria from hypothyroid rats (Fig. 6). The difference in the rate of palmitoylcarnitine oxidation was far less (385,386). These changes may explain why both fatty acid oxidation and fatty acid synthesis can show increased rates in the liver of thyrotoxic rats (134). A different picture is found in rats fed the drug clofibrate, which causes hypolipidemia. This drug induces both an increased mitochondrial mass and October 1983 CARNITINE-METABOLISM AND 1455 FUNCTIONS 8 5 25 Malonyl-CoA (FM) FIG. 6. Effect of fasting, of thyroid state, and of malonyl-CoA on carnitine-dependent oxidation of palmitoyl-CoA in isolated rat liver mitochondria. [1-14C]palmitoyl-CoA (70 PM), (-)carnitine (1 mM), malate (5 mM), phosphate (10 mM), and adenosine diphosphate (2 mM) were incubated in presence of 1% bovine serum albumin with liver mitochondria from fed rats (ClosecE squares), fasted rats (open squares), hypothyroid rats treated with propylthiouracil (closed c-ides), hypothyroid fasted rats (open cirdes), and hyperthyroid rats treated with triiodothyronine (open triangles). Oxidation was measured as perchloric acid-soluble radioactivity. Bars represent standard deviation of results obtained with 3-5 different mitochondrial preparations. increased activities of carnitine acetyltransferase and carnitine palmitoyltransferase in liver. The acetyltransferase in particular shows a great increase in activity (lo-400fold) (219,382), but it is mainly the inner carnitine acyltransferases that show increased activity (381). In addition clofibrate induces increased activity of the peroxisomal P-oxidation and chain shortening of fatty acids and of peroxisomal carnitine acetyltransferase and medium-chain acyltransferase (31, 74, 237). Clofibrate does not affect the malonyl-CoA sensitivity of the palmitoyltransferase (386). Thus the oxidation of fatty acids in the liver of clofibrate-treated animals is stimulated by mechanisms different from those of fasted or thyrotoxic rats. The carnitine translocase has also been found to increase its activity in the livers of fasted, diabetic, and clofibrate-fed rats (325, 329) and possibly also after treatment with glucagon (443). Researchers have not yet determined the mechanisms by which fasting Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 0 1456 JON BREMER Vohww and hormones change the latency and the malonyl-CoA sensitivity carnitine acyltransferase as well as the activity of the translocase. C. Allover 63 of the Regulation XVIII. FATTY Carnitine ACID OXIDATION palmitoyltransferase (181, 302, 353), as are carnitine and long-chain IN acylcarnitines EXTRAHEPATIC TISSUES is present in all animal and human tissues acetyltransferase, carnitine, acetylcarnitine, (43,264,333). Variations in the tissue contents Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 These different studies show that the rate of fatty acid oxidation in the liver is probably regulated by a series of mechanisms. Lipolysis in adipose tissues is of primary importance because it regulates the availability of free fatty acids to the liver. Inhibition of the outer carnitine palmitoyltransferase by malonyl-CoA probably helps to shut off fatty acid oxidation under lipogenic conditions. A variable rate of triacylglycerol synthesis may trap fatty acids, and the fatty acid-binding protein may direct fatty acids to esterification. Pyruvate and extramitochondrial NADH products of glycolysis (and activation of the pyruvate dehydrogenase and of the malate/aspartate shuttle) may compete with acylcarnitines in the mitochondria, particularly when acylcarnitine levels are low. Along with these extramitochondrial changes, the mitochondrial carnitine-dependent transfer mechanism for fatty acids seems to change-it is activated by fasting, by thyroid hormones, and possibly by other factors (clofibrate). Such changes in the mitochondrial transfer system itself are still poorly understood. It is striking that the outer palmitoyltransferase and the carnitine translocase change their activities in the same direction in the liver. At present it is assumed that the outer transferase, the carnitine translocase, and the inner transferase all are separate (conjugated?) units. Perhaps a carnitine acyltransferase in the membrane catalyzes all the reactions presently supposed to be the functions of three protein molecules. If this were the case, one must again consider the idea of a vectorial enzyme in the membrane (439). It is necessary, however, to interpret observations on isolated mitochondria with caution. Siess et al. (370) have recently shown that treatment of rats with glucagon may stabilize rather than activate different mitochondrial functions during the subsequent isolation procedures. Activation or inhibition of phospholipases in the membranes may be involved. Treatment of the animals with other agents may provoke similar phenomena. Further work may show whether the observed changes in liver mitochondria correspond to events in the intact cell. October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1457 A. Heart and Skeletal Muscle Owing to the large muscle mass, fatty acid oxidation in muscle tissue is quantitatively important. Muscle tissue is responsible for the oxidation of a major fraction of circulating fatty acids (188). Oxidation of fatty acids in skeletal muscle and heart is mainly controlled, by the supply of fatty acids (i.e., by the concentration of free fatty acids in the circulation) but also by work load (86, 314). Training increases the capacity of muscle tissue to utilize fatty acids, and an integrated part of this adaptation is induction of an increased mitochondrial mass and an increased activity of carnitine palmitoyltransferase (283). Conversely inactivation of the muscle by denervation leads to a drop in the activity of both carnitine palmitoyltransferase and carnitine acetyltransferase. Carnitine is also lost from denervated muscle (225). In the heart -85% of the tissue content of CoA is found in the mitochondria, whereas 90% of the carnitine is outside the mitochondria (315). These distributions are likely to cause activated fatty acids to be funneled toward oxidation rather than toward lipid synthesis. This reasoning is supported by observations on pathological conditions associated with carnitinedepleted muscle tissue. In such conditions intracellular accumulation of triacylglycerol is usually found (see sect. XXII; 78, 142). In muscle tissues, which lack the ketogenesis occurring in liver and kidney, ,&oxidation of fatty acids must be rigidly coupled to the citric acid cycle through the common mitochondrial pool of CoA, especially under conditions in which, for example, the heart uses fatty acids almost exclusively for its energy needs. Feedback mechanisms must exist by which a variable use of acetyl-CoA in the mitochondria (due to a variable work load) can Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 of long-chain acylcarnitines with the nutritional state of the animal show that carnitine most likely is involved in regulation of fatty acid oxidation in all tissues (43,333). This assumption is supported by the observation that carnitine palmitoyltransferase is inhibited by malonyl-CoA in all tissues tested: liver, kidney, adipose tissue, mammary gland, skeletal muscle, heart, and brown adipose tissue (353,355). Less is known about the distribution of malonyl-CoA in the tissues, but recently we have found that, besides the liver, both heart and kidney have measurable levels of malonyl-CoA and that these levels decrease in fasting (B. Singh, J. Bremer, and B. Borrebak, unpublished results). The regulation of fatty acid oxidation has been studied mainly in liver, heart, and brown adipose tissue and to some extent in skeletal muscle. Carnitine-dependent fatty acid oxidation has also been directly demonstrated in a series of other tissues, however, such as adipose tissue (186), salivary gland (ZOO), kidney (62), placenta (394), adrenals (184), sperm (207), and probably brain (61, 62). 1458 JONBREMER v&mw 63 B. Brown Adipose Tissue Brown adipose tissue chondrial system for fatty has a highly active, carnitine-dependent mitoacid oxidation (192). Mitochondrial metabolism Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 adjust the rate of fatty acid uptake, activation, and acylcarnitine formation in the extramitochondrial compartment. Neely and co-workers (210,314,315) have suggested that such a regulatory system exists in a coupling of the two compartments through carnitine acetyltransferase. From studies on perfused rat hearts with variable work loads, they have shown that the acetyl-CoA/ CoA ratio in the mitochondria is probably in equilibrium with the extramitochondrial acetylcarnitine/carnitine ratio and possibly with the extramitochondrial acetyl-CoA/CoA ratio. Thus an increase in the mitochondrial acetyl-CoA/CoA ratio will adjust the extramitochondrial pools of free CoA and free carnitine available for fatty acid activation and acylcarnitine formation. The carnitine palmitoyltransferase of muscle tissues is more sensitive to malonyl-CoA than, for example, the liver enzyme (353); the carnitine acetyltransferase is completely insensitive (254). Recently we have found that the heart contains measurable amounts of malonyl-CoA that decrease in fasting. Thus malonyl-CoA probably modifies the regulatory mechanism mentioned above (e.g., when pyruvate is available for oxidation). In this connection, note that fatty acid oxidation in the heart is inhibited when the (267). Acetyl-CoA oxidation of pyruvate is stimulated with dichloroacetate and acetylcarnitine increase concomitantly. Fatty acid oxidation in muscle may also be influenced by feedback inhibitions through the P-oxidation chain. Acetyl-CoA is a product inhibitor of the acetyl-CoA acyltransferase (311), and ,&ketoacyl-CoA is an inhibitor of the acyl-CoA dehydrogenase (131, 359). Long-chain acyl-CoA and long-chain acylcarnitine accumulate in the anoxic heart, whereas the levels of acetyl-CoA and acetylcarnitine decrease, probably because accumulation of NADH and reduced flavoproteins in the mitochondria inhibits fatty acid oxidation (368, 422). Thus the regulation of fatty acid oxidation in muscle tissues appears to be a complicated interplay of the availabilities of fatty acids, pyruvate, and lactate; the acetyl-CoA/CoA and acetylcarnitine/carnitine ratios; the level of malonyl-CoA; and the redox situation in the mitochondria. Palmitoylcarnitine is a relatively strong inhibitor of the Na+-K+ATPase, and it has been suggested that the accumulation of long-chain acylcarnitine in the ischemic heart explains the loss of K+ from heart in ischemia (434). Palmitoylcarnitine also inhibits phospholipid-sensitive protein kinases (3). It is still uncertain whether these effects represent more than unspecific detergent effects or whether they represent physiological regulatory mechanisms. October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1459 XIX. CARNITINE ACYLTRANSFERASES IN NEWBORNS In the newborn a switch from carbohydrate to fatty acid oxidation for energy takes place. In fetal liver and heart the rate of fatty acid oxidation is low (6, 418, 419, 427). Immediately after birth a rapid increase in the activity of carnitine palmitoyltransferase and in the capacity to oxidize fatty acids takes place in liver (6, 32) and in heart (20, 418). An increase in the activity of the acetyltransferases also takes place in different tissues after birth (177, 238,417). In the chick embryo a similar increase in the activities of the carnitine acyltransferase takes place a few days before hatching (94, 230). In the fetal heart it is the outer carnitine palmitoyltransferase in particular that shows a low activity, whereas the inner transferase is more “normal.” Thus although fetal calf heart mitochondria oxidize palmitoylCoA only at a slow rate in the presence of carnitine, palmitoylcarnitine is rapidly oxidized. Shortly after birth palmitoyl-CoA is also rapidly oxidized (419). The outer transferase evidently is present in a latent form. Preincubation of isolated mitochondria on ice for 2 h (82), incubation of mitochondria Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 in this tissue is unusual in that the mitochondria physiologically can operate reversibly in a loosely coupled state (113, 295). This is the mechanism for nonshivering thermogenesis. Fatty acids made available by a catecholamine-triggered lipolysis seem to regulate the rate of fatty acid oxidation (336). The reversible uncoupling is regulated by a specific translocase protein that renders the inner mitochondrial membrane permeable to hydrogen ions (294). This translocase is inactivated by GTP, which binds to the translocase, and it is probably activated by free fatty acids (248). Thus the free fatty acids trigger both acylcarnitine formation and uncoupling, which together make rapid heat production possible. In the brown adipose tissue of the hamster, which is a true hibernator, free acetate can be the end product of acylcarnitine oxidation. This is due to the presence of an acetyl-CoA hydrolase in the mitochondria (25,26). An interesting feature of this system is that the &oxidation of palmitoylcarnitine to acetate is as rapid at 10°C as at 37”C, whereas the citric acid cycle cannot function at 10°C. Hence the oxidation to free acetate may represent an initial “ignition” in the early heating phase of this hibernator. Even in the cold-adapted rat, brown adipose tissue is very active in fatty acid synthesis (268, 405). Thus malonyl-CoA must be formed. The outer carnitine palmitoyltransferase in this tissue is strongly inhibited by malonyl-CoA, even in the cold-adapted state (355). This may seem paradoxical in a tissue geared to heat production through fatty acid oxidation, and an interesting problem is how catecholamine-induced lipolysis, uncoupling, and malonyl-CoA inhibition interact in the regulation of fatty acid oxidation in this tissue. 1460 JON BREMER Vokwrne 63 XX. CARNITINE AND FERTILITY In 1965 Marquis and Fritz (264) found that epididymis contains an extremely high concentration of carnitine and that spermatozoa have a >high activity of carnitine acetyltransferase. They also showed that this high concentration and high enzyme activity depend on an effect of testosterone. These observations led to extensive studies on the function of carnitine in spermatogenesis and fertility. The development of the high activity of carnitine acetyltransferase in the spermatozoa coincides with the maturation of sperm cells (416). As they pass through the epididymis, the sperm cells take up relatively large amounts of carnitine from the epididymal fluid, which contains up to 60 mM carnitine. By the time the spermatozoa reach the cauda epididymidis they have lost the ability to take up carnitine, but they now contain high concentrations of carnitine and acetylcarnitine and a high carnitine acetyltransferase activity (80,91,92). A similar development takes place in the spermatozoe of Drosophila mlanogaster (165). The spermatozoa have the ability to utilize a series of substrates for energy. During transit through the epididymis the oxidation of fatty acids seems to be important. These fatty acids may be derived from the breakdown of phospholipids (187), and their oxidation is carnitine dependent (90). In this connection it is interesting that the epididymis contains an androgeninduced phospholipase (34) and that the epididymal fluid contains high concentrations of glycerophosphocholine (80). In mature ejaculated sperm, however, fatty acid oxidation seems to be relatively unimportant, and the fertilizing capacity of the spermatozoa seems to depend on glycolysis of fructose and glucose and on oxidation of lactate and pyruvate (93, 207, 278, 391). Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 in a medium with KC1 instead of sucrose, preincubation of mitochondria with palmitoyl-CoA and carnitine (in the absence of adenosine diphosphate; 403), or pretreatment of mitochondria with phospholipase C (EC 3.1.4.3) (433) all lead to exposure of latent palmitoyltransferase and more rapid oxidation of palmitoyl-CoA in the presence of carnitine. It is striking that mitochondria isolated from adult ischemic dog hearts show a low activity of outer carnitine palmitoyltransferase similar to that of fetal mitochondria (435). In this case a decrease in the K, for carnitine was also found, and the authors have suggested that lipid changes in the membrane, which affect the hydrophobic region of the carnitine palmitoylmight cause the observed phenomena. transferase (see sect. XIV@, The variable latency of the heart carnitine palmitoyltransferase may be a phenomenon similar to the variable latency of the same enzyme in liver; more enzyme seems to be exposed on the surface of the inner mitochondrial membrane in the livers of animals after fasting (48, 67) or after treatment 385, 386). Fasting of the animal with 3,5,3’-triiodothyronine (see sect. XVIIB; has no effect, however, on the heart enzyme (385). October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1461 XXI. CARNITINE AND BRANCHED-CHAIN AMINO ACID METABOLISM When isolated mitochondria are incubated with branched-chain cu-keto acids and carnitine, branched-chain acylcarnitines are formed (383). Acetyl-, propionyl-, and butyrylcarnitine are formed with pyruvate, a-ketobutyrate, and a-ketopentanoate (66, 303). Thus mitochondria contain carnitine acyltransferase(s) able to transfer both straight-chain and branched-chain acyl groups to carnitine. Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 In mature spermatozoa the relation of carnitine and carnitine acetyltransferase to pyruvate metabolism is similar to that found in other tissues. When sperm is incubated with pyruvate or with pyruvate precursors such as lactate, glucose, or fructose, a rapid oxidation of pyruvate takes place. At the same time the endogenous carnitine is acetylated by the carnitine acetyltransferase and by the acetyl-CoA formed from pyruvate. The acetylcarnitine level remains high as long as pyruvate is available (93,207,278). When pyruvate is exhausted, the stored acetyl groups are transferred back to CoA and oxidized in the citric acid cycle. This is similar to what takes place in mitochondria from heart (61) and from blowfly flight muscle (109). Mature spermatocytes contain over 1,000 times more carnitine than CoA. Thus sperm can accumulate significant amounts of easily available activated acetate [up to 2-3 pmol/lO’ cells, or 150 pmol/g dry matter (93)]. In the absence of external substrates the oxidation of endogenous acetylcarnitine in the citric acid cycle can account for the entire oxygen uptake in bovine spermatozoa. Even when sperm motility is stimulated by cyclic adenosine monophosphate after addition of caffeine, the acetylcarnitine can support-respiration for several minutes (278). Because the cell membrane in mature sperm is impermeable to acetylcarnitine (91), this substrate is not lost by diffusion in a substrate-free environment. In this connection it is noteworthy that the motility of ejaculated sperm (one of the indexes of their fertilizing capacity) correlates with their acetylcarnitine content (216). Perhaps carnitine has specific functions in fertility other than storing acetyl groups. High concentrations of external carnitine, such as those found in the epididymis, suppress respiration in mature bovine spermatozoa, and this might have a function while the spermatozoa are stored in the cauda epididymidis (180). This effect of carnitine seems to be connected with its binding to the cell membrane. The effect is observed only with ejaculated bovine sperm, however, not with spermatozoa taken from the cauda epididymidis. The effect is also absent from human sperm (215). Tanphaichitr (395) reported that external acetylcarnitine stimulates motility in human sperm. The nature of this effect is not clear. In summary, only its function as a store for activated acetyl groups has been established as a general function of carnitine in spermatogenesis and fertility. 1462 JON BREMER Volume 63 Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 After perfusion of rat hearts and rat hindquarters with branched-chain cu-keto acids or with a)-ketobutyrate, substantial amounts of the same acylcarnitines are formed in the muscle tissues (132,384). Even in normal intact rats small amounts of propionylcarnitine (37, 40) and branched-chain acylcarnitines can be identified in the tissues (30, 110). In sheep liver there may even be more propionylcarnitine than acetylcarnitine (375). In isolated heart and muscle mitochondria and in muscle homogenates carnitine stimulates the oxidation of branched-chain cu-keto acids (70, 331, 414), and the corresponding branched acylcarnitines inhibit such oxidation (168, 413). Fatty acid esters of carnitine also inhibit the oxidation of cu-keto acids (70). On the other hand, isolated mitochondria oxidize isobutyrylcarnitine (111). These different observations have led to speculations that carnitine funtions specifically in the catabolism of branched-chain amino acids by permitting transport into the mitochondria of the activated branched-chain carboxylic acids, which are formed by the branched-chain cu-keto acid dehydrogenase. This idea was reasonable because the mitochondrial branchedchain ar-keto acid dehydrogenase was originally reported to be located on the outer surface of the inner mitochondrial membrane (218). Isolated mitochondria can decarboxylate branched-chain cu-keto acids in the absence of carnitine, however, and also in the absence of external CoA and NAD, which are cofactors in the reaction (70, 266). The cu-keto acid dehydrogenase (as pyruvate dehydrogenase) is therefore located inside the CoA permeation barrier, presumably in the matrix. The formation of branched-chain acylcarnitines thus seems to be a “dead-end” reaction, because the carnitine esters must be transferred back into the mitochondria for further metabolism. Because small amounts of free branched-chain carboxylic acids are found in blood (8), Van Hinsbergh and colleagues (413) suggested that carnitine has a function in the release of these carboxylic acids from muscle tissue. Mitochondria contain acyl-CoA hydrolases (22, 49), however, whereas only a high-K, acylcarnitine hydrolase has been found extramitochondrially (21, 259). Hence carnitine probably does not play a major role in the release of these carboxylic acids. At present it is certain only that the presence of extramitochondrial propionyl and branched-chain acylcarnitines in the tissues mirrors the intramitochondrial acyl-CoA situation, because the short-chain carnitine acyltransferase(s) has a relatively broad acyl group specificity. This formation of branched-chain acylcarnitines by the mitochondria, like the formation of acetylcarnitine, may be part of a general buffering effect of carnitine on the mitochondrial acyl-CoA/CoA ratios, preventing “flooding” of the mitochondrial matrix by CoA esters. The level of propionyl and branched-chain acylcarnitines in the tissues may be a metabolic indicator of the catabolic rate of branched-chain amino acids. October XXII. 1983 CARNITINE CARNITINE-METABOLISM IN PATHOGENIC AND FUNCTIONS 1463 MECHANISMS A. Loss and Lack of Car&tine Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 With the key role of carnitine in fatty acid oxidation in mind, it is not surprising that disturbances in the function of carnitine may cause pathological phenomena. When carnitine is lacking, a common feature seems to be the accumulation of triacylglycerol in the cell; under these conditions an inhibited fatty acid oxidation diverts fatty acids from oxidation to esterification (lipidosis). Wittels and Bressler (78, 426) reported the first example of such a condition. They found that the well-known lipidosis and heart failure in diphtheria may be explained by a loss of carnitine from the heart. In guinea pigs the administration of the toxin of Neisseria diphtheria induced lipidosis of the heart, and homogenates of these hearts had a decreased ability to oxidize fatty acids. The addition of carnitine normalized the oxiof carnitine to diphtheritic animals in vivo dation rate (78). Administration has a protective effect against the toxin and prolongs the survival of the animals (98, 99). Malstad and Brshmer (286) studied the effect of the toxic in tissue culture and suggested that the cells lose carnitine because the toxin, which is an inhibitor of protein synthesis, prevents the synthesis of the carnitine carrier in the cell membrane. Prednisolone in the medium had a protective effectpresumably it slowed the breakdown of the carrier (285). This effect of prednisolone may be the basis for its beneficial effect in hereditary carnitine deficiency (see below; 212, 412). Loss of carnitine may also contribute to heart failure in other conditions. In experimental heart failure produced by constriction of the aorta, heart carnitine decreases (429), as it does in severe ischemia of the heart (364, 368). In severe liver cirrhosis the carnitine content in plasma and in different tissues was found to be only one-fourth to one-third of normal, and urinary excretion of carnitine was even more reduced (349). Both spontaneous dietary intake and biosynthesis of carnitine appeared to be far below normal in these patients. Carnitine deficiency also may be an iatrogenic condition. In kidney patients on hemodialysis great amounts of carnitine are lost in the dialysis fluid, and the carnitine content in plasma and muscle is often low (12, 39). This is believed to contribute to the hypertriglyceridemia, cardiomyopathy, and cardiac failure seen in these patients. Substitution therapy wth carnitine has been found to diminish hypertriglyceridemia (172, 339). Plasma carnitine is often elevated in untreated renal insufficiency (44, 108), presumably because of a lowered clearance in the kidneys. Carnitine deficiency may also be provoked in infants given artificial carnitine-free diets, presumably because of a slow endogenous rate of carnitine 1464 JON BREMER Vohm 63 biosynthesis in the neonatal period (373). Newborn infants normally receive substantial amounts of carnitine from their mothers’ milk (53). Disturbed carnitine metabolism has also been suggested in other clinical conditions, although its significance seems uncertain (281). B. I- Errors of Carnitine Metabolism Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 In 1973 the first two cases of inborn errors of carnitine metabolism were reported. Engel and Angelini (142) found a case of lipid-storage myopathy associated with muscle fatigue and a low carnitine content in skeletal muscle, and DiMauro and DiMauro (136) reported a case in which muscle cramps and myoglobinuria after exercise were associated with a low activity of carnitine palmitoyltransferase in muscle, whereas the muscle carnitine level was normal. Subsequently a series of similar cases of varying severity has been reported in the literature. DiMauro and co-workers (137) have reviewed these and other disorders of lipid metabolism in muscle. Carnitine deficiency may vary in severity. In some cases the condition seems to be limited to skeletal muscle-the serum carnitine and the carnitine contents in other tissues are normal. In some cases the carnitine deficiency is general (systemic carnitine deficiency), with low carnitine concentrations in plasma and in all tissues. Several fatal cases have been reported (100,125, 221,412). The age when symptoms appear may range from early childhood for systemic cases to late adolescence for cases limited to muscle. In the less severe cases a normal development of ketonemia in fasting has been observed (thus showing a normal ability of the liver to utilize fatty acids), whereas the systemic cases have a reduced or absent fasting ketonemia. A corresponding decrease in carnitine content in the liver has been found (135). It has been assumed that the isolated muscle deficiency is due to defective transport of carnitine into the tissue, because the plasma carnitine is normal (412). In accordance with this view, the addition of carnitine to muscle homogenates gave normal fatty acid oxidation (142), although in another case no such normalization was obtained (424). In systemic carnitine deficiency a defect in carnitine biosynthesis has been suggested. However, a normal conversion of &V-trimethyl+lysine to to carnitine was found in three patients (341, 343). In one case a defect in the conversion of y-butyrobetaine to carnitine was found (C. Hoppel, personal communication). Although a moderately decreased reabsorption of carnitine in the kidneys and an increased clearance of carnitine were found in one study of systemic carnitine deficiency, this defect was also found in a control with normal carnitine in the tissues (143). The investigators therefore concluded that the renal abnormality could at most only contribute to the carnitine deficiency in the tissues. Possibly a general defect in carnitine transport in the tissues (including the kidneys and gut) could explain the general deficiency. The absorption of carnitine in the gut has not been systematically October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1465 Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 investigated, either in normal persons or in those with carnitine deficiency. In some cases of carnitine deficiency, however, patients have responded favorably to carnitine substitution therapy. The plasma carnitine was restored to normal, showing that carnitine is absorbed, but muscle content was not restored (100). So far it is not established with certainty whether muscle and systemic carnitine deficiencies represent different disease entities or whether they represent varying severities of the same disease in different stages of development. In one case of muscle deficiency VanDyke and co-workers (412) found moderately lowered carnitine contents in the skeletal muscles of a patient’s parents, both of whom were clinically. normal. Consanguinity of the parents could be traced in another study of two affected sisters (424). These and other cases suggest therefore that the disease is inherited as an autosomal recessive disorder. In systemic carnitine deficiency increased amounts of dicarboxylic acids are found in the urine. Electron-microscopic investigation of the liver showed proliferation of the endoplasmic reticulum and an increased number of peroxisomes (221). These observations suggest that the inhibited mitochondrial oxidation of fatty acids leads to increased w-oxidation of fatty acids and to peroxisome proliferation and increased peroxisomal P-oxidation, which is carnitine independent (118). Apparently the auxiliary @oxidation system of the peroxisomes and the o-oxidation system of the endoplasmic reticulum represent adaptive systems that are stimulated when the mitochondrial carnitine-dependent P-oxidation system shows a (relative) insufficiency. This may occur in a number of situations: in starvation and diabetes (35), in feeding of high-fat diets (292, 401), or when mitochondrial fatty acid oxidation is inhibited by poorly oxidizable fatty acids (74), by inhibitors (46), or by suboptimal carnitine concentrations. At least 15 cases of carnitine palmitoyltransferase deficiency of different severities have been reported (214). The patients suffer from muscle cramps and myoglobinuria, especially after prolonged exercise, and the condition is aggravated by fasting and/or high-fat diets. The carnitine palmitoyltransferase activity in skeletal muscle has been found to range from almost none to nearly 50% of normal. Those with severe deficiency experience symptoms early in childhood, whereas those with moderately depressed enzyme activity experience their first symptoms as teenagers after prolonged exercise. The severe cases also have a depressed ketone body production in fasting, showing that the liver may also be affected. This has been confirmed in one case by direct measurement of enzyme activity in the liver (27). In other cases leukocytes, thrombocytes, and fibroblasts have reduced levels of the enzyme. Thus the disease most likely is general, with symptoms primarily in the muscles. In two cases carnitine acetyltransferase was also measured and was found to be reduced, but to a lesser extent than palmitoyltransferase (136, 189). 1466 JON BREMER vohme 63 XXIII. SUMMARY Carnitine was detected at the beginning of this century, but it was nearly forgotten among biochemists until its importance in fatty acid metabolism was established 50 years later. In the last 30 years, interest in the metabolism and functions of carnitine has steadily increased. Carnitine is synthesized in most eucaryotic organisms, although a few insects (and most likely some newborn animals) require it as a nutritional factor (vitamin BT). Carnitine biosynthesis is initiated by methylation of lysine. The trimethyllysine formed is subsequently converted to butyrobetaine in all tissues; the butyrobetaine is finally hydroxylated to carnitine in the liver and, in some animals, in the kidneys (see Fig. 1). It is released from these tissues and is then actively taken up by all other tissues. The turnover of carnitine in the body is slow, and the regulation of its synthesis is still incompletely understood. Microorganisms (e.g., in the intestine) can metabolize carnitine to trimethylamine, dehydrocarnitine (@keto-y-trimethylaminobutyric acid), betaine, and possibly to trimethylaminoacetone. In some insects carnitine can be converted to methylcholine, presumably with trimethylaminoacetone as an intermediate(see Fig. 3). In mammals the unphysiological isomer (+)carnitine is converted to trimethylaminoacetone. The natural isomer (-)car- Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 Most patients with carnitine palmitoyltransferase deficiency have no significant accumulation of triacylglycerol in their muscle cells, despite a normal content of long-chain acyl-CoA synthase. They do, however, have elevated plasma triacylglycerol levels (10, 136, 361). Thus fatty acid uptake in the tissues seems to be inhibited. This difference from muscle carnitine deficiency is not understood. In some cases both the outer and inner carnitine palmitoyltransferases showed reduced activity (136); in two cases, however, the defect has been assumed to be limited to the inner transferase (330, 361) and in one case to the outer transferase (198). In the case where the deficiency was localized to the outer transferase, isolated muscle mitochondria oxidized palmitoylcarnitine but showed no activity with palmitoyl-CoA plus carnitine. Because of these differences some researchers have proposed that the outer and inner transferases are under different genetic controls (361). Others have suggested that the outer and the inner transferases are one and the same enzyme (24). In most cases it has been assumed that the disease is inherited as an autosomal disorder. A family with a combined deficiency of carnitine (40% of normal) and of carnitine palmitoyltransferase (SO-65% of normal) has also been reported. The family had six members in four generations with similar symptoms (muscle cramps and dark urine), suggesting an autosomal dominant inheritance (211). October 1983 CARNITINE-METABOLISM AND FUNCTIONS 1467 REFERENCES 1. AAS, M. Organ and subcellular distribution of fatty acid activating enzymes in the rat. Biochim Bicphys AC&Z 231: 32-47, 1971. 2. AAS, M., AND J. BREMER. Short-chain fatty acid activation in rat liver. A new assay procedure for the enzymes and studies on their intracellular localization. Biochim. Biophys Actu 164: 157-166, 1968. 3. ADAMS, R. J., D. W. COHEN, S. GUPTE, J. D. JOHNSON, E. T. WALLICK, T. WANG, AND A. SCHWARTZ. In vitro effects of palmitylcarnitine on cardiac plasma membrane Na,K-ATPase, and sarcoplasmic reticulum Ca*+-ATPase and Ca*+ transport. J. Biol Chem 254: 12404-12410, 1979. 4. ALKONYI, I., J. KERNER, AND A. SkNDOR. The pos- Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on October 21, 2016 nitine is excreted unchanged in the urine, and it is still uncertain if it is degraded in mammalian tissues at all (Fig. 2). The only firmly established function of carnitine is its function as a carrier of activated fatty acids and activated acetate across the inner mitochondrial membrane. Two acyl-CoA:carnitine acyltransferases with overlapping chain-length specificities have been isolated: one acetyltransferase taking part in the transport of acetyl and short-chain acyl groups and one taking part i.n th e transport of ’ long- chain acyl groups. Pal mitoyltransferase An additional octanoyltransferase has been isolated from liver peroxisomes. Although a carnitine translocase that allows carnitine and acylcarnitine membrane has been deduced from functo penetrate the inner mitochondrial tional studies (see Fig. 5), this translocase has not been isolated as a protein separate from the acyltransferases. Carnitine acetyltransferase and carnitine octanoyltransferase are also found i n the peroxisomes. In these organelles the enzymes m .ay be important in the transfer of acyl groups, which are produced by the peroxisomal poxidation enzymes, to the mitochondria for oxidation in the citric acid cycle. The carnitine-dependent transport of activated fatty acids across the mitochondrial membrane is a regulated process. Malonyl-CoA inhibits this process in all tissues. In liver the activity of the outer palmitoyltransferase in the membrane appears to be regulated by the nutritional state of the animal and by hormones (3,5,3’-triiodothyronine). Its sensitivity to malonylCoA also seems to vary with nutritional state (Fig. 6). The nature of these potentially important regulatory mechanisms is still unknown and represents a challenge for further research. Inborn errors of carnitine metabolism and function are known. In some patients the activity of carnitine palmitoyltransferase is decreased, and they suffer from muscle fatigue an .d myoglobi nuria. In severe cases the liver has a reduced capacity to oxidi .ze fatty acids to ketone bodies. Carnitine deficiency in tissues is also a recognized disorder. In some cases the condition is limited to skeletal muscle. Patients having this deficiency suffer from muscle fatigue and muscle lipidosis. In the systemic deficiency all tissues seem to lack carnitine, and fatal cases have been reported. The cause may be an inability of the tissues to concentrate and retain carnitine. Conditions are also recognized in which the loss of carnitine from tissues (or the lack of carnitine in the diet) is part of pathogenic mechanisms. 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