Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.     

Cardiac carnitine deficiency and altered carnitine transport in cardiomyopathic hamsters

The Bio 14.6 hamster has a well-documented cardiomyopathy which leads to congestive heart failure. Previous work demonstrated that hearts from these hamsters have depressed fatty acid oxidation and depressed carnitine concentrations compared to those of normal hamsters. Analyses of tissue carnitine concentrations from 40 to 464 days of age demonstrate that the cardiomyopathic hamsters have a cardiac carnitine deficiency throughout life. Therefore, the carnitine deficiency is not a secondary effect of an advanced stage of the cardiomyopathy. Both the observation that other tissues of the cardiomyopathic hamster have normal or markedly elevated carnitine concentrations and the observation that oral carnitine treatment could not increase the cardiac carnitine concentrations to those of normal hamsters are consistent with the hypothesis that the cardiac carnitine deficiency is the result of a defective cardiac transport mechanism. Cardiac carnitine-binding protein (which may function in the cardiac carnitine transport mechanism) prepared from hearts of cardiomyopathic hamsters had a lower maximal carnitine binding and an increased dissociation constant for carnitine compared to the cardiac carnitine-binding protein prepared from normal hamsters. Thus, several types of data indicate that the cardiomyopathic hamster has an altered cardiac carnitine transport mechanism.     

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Influence of carnitine supplementation on muscle substrate and carnitine metabolism during exercise

We examined 1) the effect of L-carnitine supplementation on free fatty acid (FFA) utilization during exercise and 2) exercise-induced alterations in plasma levels and skeletal muscle exchange of carnitine. Seven moderately trained human male subjects serving as their own controls participated in two bicycle exercise sessions (120 min, 50% of VO2max). The second exercise was preceded by 5 days of oral carnitine supplementation (CS; 5 g daily). Despite a doubling of plasma carnitine levels, with CS, there were no effects on exercise-induced changes in arterial levels and turnover of FFA, the relation between leg FFA inflow and FFA uptake, or the leg exchange of other substrates. Heart rate during exercise after CS decreased 7-8%, but O2 uptake was unchanged. Exercise before CS induced a fall from 33.4 +/- 1.6 to 30.8 +/- 1.0 (SE) mumol/l in free plasma carnitine despite a release (2.5 +/- 0.9 mumol/min) from the leg. Simultaneously, acylated plasma carnitine rose from 5.0 +/- 1.0 to 14.2 +/- 1.4 mumol/l, with no evidence of leg release. Consequently, total plasma carnitine increased. We concluded that in healthy subjects CS does not influence muscle substrate utilization either at rest or during prolonged exercise and that free carnitine released from muscle during exercise is presumably acylated in the liver and released to plasma.     

Myocardial carnitine and carnitine palmitoyltransferase deficiencies in patients with severe heart failure

We studied myocardial tissue from 25 cardiac transplant recipients, who had end-stage congestive heart failure (CHF), and from 21 control donor hearts. Concentrations of total carnitine (TC), free carnitine (FC), short-chain acylcarnitines, long-chain acylcarnitines (LCAC) as well as carnitine palmitoyltransferase (CPT) activities were measured in myocardial tissue homogenates and referred to the concentration of non-collagen protein. Compared to controls, the concentrations of TC and FC as well as total CPT activities were significantly lower in patients. LCAC levels and the LCAC to FC ratio values were significantly greater in patients than in controls. While the malonyl-CoA sensitive fraction of CPT, which represents CPT I activity, was similar in patients and controls, the residual CPT activity after inhibition by malonyl-CoA, representing CPT II activity, was significantly reduced in patients compared to controls. Moreover, the activity of CPT in the presence of Triton X-100, which also represents the activity of CPT II, was significantly lower in patients than in controls. Malonyl-CoA concentrations required for half-maximal inhibition of CPT activity were significantly greater in patients than in controls. There was a linear relationship between ejection fraction (EF) values and concentrations of TC, FC, or total CPT activities. Values for LCAC and the LCAC to FC ratio were inversely related to EF values. We conclude that failing heart shows decreased total CPT and CPT II activities and carnitine deficiency that may be related to ventricle function.     

The sidedness of carnitine acetyltransferase and carnitine octanoyltransferase of rat liver endoplasmic reticulum

The location of carnitine acetyltransferase and carnitine octanoyltransferase on the inner and outer surfaces of rat liver microsomes was investigated. Latency of mannose-6-phosphate phosphatase showed that the microsomes were 90–94% sealed. All of the octanoyltransferase is associated with the cytosolic face, while the acetyltransferase is distributed between the cytosolic face (68–73%) and the lumen face (27–32%) of the endoplasmic reticulum membrane. Small amounts of trypsin inhibit the carnitine octanoyltransferase equally in either sealed or permeable microsomes but the acetyltransferase of sealed microsomes is stimulated. Large amounts of trypsin inhibit all transferase activities by about 60%, except for acetyltransferase of sealed microsomes. Other studies show that 0.1% Triton X-100 partially inhibits carnitine octanoyltransferase of microsomes but does not inhibit the acetyltransferase or any of the mitochondrial carnitine acyltransferase.     

Stereoisomeric acylamidomorpholinium carnitine analogues: selective inhibitors of carnitine palmitoyltransferase I and II

Acylamidomorpholinium carnitine analogues, 6-(tetradecanamidomethyl- and -hexadecanamidomethyl)-4,4-dimethylmorpholin-4-ium-2-a cetate, 1, synthesized as complete sets of stereoisomers, were assayed as inhibitors for isozymes of carnitine palmitoyltransferase (CPT). Microsomal CPT isoymes showed modest discrimination among the stereoisomers; while rat-liver mitochondrial CPT-I and CPT-II showed distinct differences. The tetradecanamidomethyl analogue of (2R,6S)-1 activated CPT-I but inhibited CPT-II.     

Stereopharmacology of carnitine

L-Carnitine (L-beta-hydroxy-gamma-N,N,N-trimethylaminobutyric acid) plays an essential role in fatty acid transport in the mitochondrion. Conditions that appear to benefit from exogenous supplementation of L-carnitine include anorexia, chronic fatigue, cardiovascular disease, hypoglycemia, male infertility, muscular myopathies, renal failure and dialysis. D-Carnitine is not biologically active and might interfere with the proper utilization of the L isomer, and so there are claims that the racemic mixture (DL-carnitine) should be avoided. Despite the fact that it is known about the systemic manifestations of oral intake of this compound, oral supplementation with DL-carnitine for treatment of primary and secondary carnitine deficiency syndromes has been used in Russia for 25 years. The purpose of the present review was to contrast the differences in pharmacokinetics, phannacodynamics, biochemistry, and toxicity between treatments of L- and DL-carnitine. There is some evidence that L-carnitine and D-carnitine compete for uptake in small intestine and tubular re-absorption in kidneys. After intestinal absorption, L- and D-carnitine is transferred to organs whose metabolism is dependent on fatty acid oxidation, such as heart and skeletal muscle, and D-carnitine competitively depletes muscle level of L-carnitine. Whereas L-carnitine is found to be essential for the oxidation of fatty acids, D-carnitine causes a depletion of L-carnitine, and hindered fatty acid oxidation and energy formation. Pharmacological effects of carnitine are stereospecific, since L-carnitine was effective in various animals and clinical studies, while D- and DL-carnitine was found to be ineffective or toxic, for example, to muscle cells and to the myocardium. DL-Carnitine causes symptoms of myasthenia and cardiac arrhythmias, which disappeared after L-carnitine administration. Clinically toxic effect of D-carnitine was described in patients with renal failure on long-term haemodialysis, in adriamycin (doxorubicin) cardiotoxicity and in stable angina pectoris.     

Effect of exogenous carnitine on carnitine homeostasis in the rat

The interaction of exogenous carnitine with whole body carnitine homeostasis was characterized in the rat. Carnitine was administered in pharmacologic doses (0-33.3 mumols/100 g body weight) by bolus, intravenous injection, and plasma, urine, liver, skeletal muscle and heart content of carnitine and acylcarnitines quantitated over a 48 h period. Pre-injection urinary carnitine excretion was circadian as excretion rates were increased 2-fold during the lights-off cycle as compared with the lights-on cycle. Following carnitine administration, there was an increase in urinary total carnitine excretion which accounted for approx. 60% of the administered carnitine at doses above 8.3 mumols/100 g body weight. Urinary acylcarnitine excretion was increased following carnitine administration in a dose-dependent fashion. During the 24 h following administration of 16.7 mumols [14C]carnitine/100 g body weight, urinary carnitine specific activity averaged only 72 +/- 4% of the injection solution specific activity. This dilution of the [14C]carnitine specific activity suggests that endogenous carnitine contributed to the increased net urinary carnitine excretion following carnitine administration. 5 min after administration of 16.7 mumol carnitine/100 g body weight approx. 80% of the injected carnitine was in the extracellular fluid compartment and 5% in the liver. Plasma, liver and soleus total carnitine contents were increased 6 h after administration of 16.7 mumols carnitine/100 g body weight. 6 h post-administration, 37% of the dose was recovered in the urine, 12% remained in the extracellular compartment, 9% was in the liver and 22% was distributed in the skeletal muscle. In liver and plasma, short chain acylcarnitine content was increased 5 min and 6 h post injection as compared with controls. Plasma, liver, skeletal muscle and heart carnitine contents were not different from control levels 48 h after carnitine administration. The results demonstrate that single, bolus administration of carnitine is effective in increasing urinary acylcarnitine elimination. While liver carnitine content is doubled for at least 6 h following carnitine administration, skeletal muscle and heart carnitine pools are only modestly perturbed following a single intravenous carnitine dose. The dilution of [14C]carnitine specific activity in the urine of treated animals suggests that tissue-blood carnitine or acylcarnitine exchange systems contribute to overall carnitine homeostasis following carnitine administration.     

Abnormal sodium stimulation of carnitine transport in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na(+)/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K(Na)) from the physiological value of 11.6 to 187 mm. Substitution of Glu(452) with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K(Na). Carnitine transport decreased exponentially with increased K(Na). The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer.     

Improved methodology to assay carnitine and levels of free and total carnitine in human plasma

Carnitine, once known as vitamin Bt, is intrinsic to human tissue and is biochemically established as being acylated with fatty acids by Acyl-CoA to give Acyl-carnitines which then are transported to the inner mitochondrial membrane by a translocase. Carnitine is of increasing clinical interest and importance, and endomyocardial deficiencies of carnitine have been reported for patients in heart failure. Consequently, a reproducible and accurate analysis of human tissue specimens for levels of free carnitine and Acyl-carnitine to guide and to support continuing clinical studies of disease states is needed. We have devised an analytical method which utilizes 5,5'-dithiobis-2-nitro-benzoate and demonstrated recovery, reproducibility and precision. Hydrolysis of a specimen at 90 degrees C for 15 min, and control of pH below 6.0 are critical steps. The mean levels of free carnitine and total carnitine in 17 ordinary subjects were 50.6 +/- 9.7 nmol./ml and 62.6 +/- 11.7 nmol./ml. respectively.     

Functional characterization of the carnitine transporter defective in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. Here we report the functional characterization of this transporter in human fibroblasts. Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake. Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency. In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+. Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine. Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters. A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range. These result indicate the presence of multiple types of carnitine transporters in human cells.     

Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.     

Purification and properties of carnitine dehydratase from Escherichia coli--a new enzyme of carnitine metabolization

Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.     

PPAR alpha-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPAR alpha-/- mice, indicating that PPAR alpha is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPAR alpha-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPAR alpha-/- mice. Here we show that both enhancement of carnitine biosynthesis (due to increased gamma-butyrobetaine dioxygenase activity), extra-hepatic gamma-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPAR alpha-dependent.