Carnitine acyltransferases and their influence on CoA pools in health and disease

Molecular Aspects of Medicine 25 (2004) 475–493 www.elsevier.com/locate/mam

Review

Carnitine acyltransferases and their influence on CoA pools in health and disease Rona R. Ramsay a

a,*

, Victor A. Zammit

b

Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, UK b Hannah Research Institute, Mauchline Road, KA6 5HL, Ayr, UK

Available online Abstract Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Carnitine; Carnitine acyltransferase; Coenzyme A; Mitochondria; Peroxisomes; Fatty acid oxidation

*

Corresponding author. Tel.: +44-1334-463411; fax: +44-1334-462595/463400. E-mail address: [email protected] (R.R. Ramsay).

0098-2997/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2004.06.002

476

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

2.

The carnitine acyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The carnitine acyltransferases––locations . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The carnitine acyltransferases––properties . . . . . . . . . . . . . . . . . . . . . . . 2.3. The carnitine acyltransferases––deficiencies . . . . . . . . . . . . . . . . . . . . . .

478 478 480 480

3.

The carnitine system protects the free CoA pools . . . . . . . . . . . . . . . . . . . . . . 3.1. Evidence for acyl interchange between carnitine and CoA pools in the cell and animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Carnitine in metabolic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Therapeutic use of short-chain acyl-carnitine esters . . . . . . . . . . . . . . . .

481 481 482 483

The influence of the carnitine system on intracellular metabolism. . . . . . . . . . . 4.1. Fuel use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Carnitine-mediated peroxisomal–mitochondrial interactions . . . . . . . . . . 4.3. Intracellular effects of therapeutic use of carnitine and its esters . . . . . . . 4.4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

485 485 486 488 490

4.

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

1. Introduction Esterification of carboxylic acids to Coenzyme A (CoA) through a thioester bond is a common strategy used in metabolic processes to ‘activate’ the relevant metabolite, generally as the first step in a pathway. The process requires an input of energy in the form of the simultaneous hydrolysis of nucleotide triphosphate. There are two universal consequences: (i) it sequesters CoA from the limited pools that exist in individual subcellular compartments, and (ii) it renders the metabolite (as its CoA ester) impermeant through cellular membranes (except when specialised membrane proteins are involved in their transfer such as in the mitochondrial (Tahiliani et al., 1992) or peroxisomal (Hettema and Tabak, 2000) membranes). As a result, the pools of CoA are maintained separate in the different cellular compartments, and may have different properties and exert separate effects in their respective locations. For example, long-chain acyl-CoA may be used not only for fatty acid oxidation in mitochondria or peroxisomes but also for complex lipid synthesis in the cytosol and endoplasmic reticulum. In the case of acyl-CoA esters, the need to control the concentration of the individual esters is imperative because of the high biological activity displayed by some of them, including the regulation of gene expression, membrane trafficking and modulation of ion-channel activities. Thus, the cell has two requirements: (i) a mechanism for the control of CoA ester concentrations that is rapid and does not involve the energetically expensive cycle of hydrolysis and

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

477

resynthesis between the esters and the free acids, and (ii) a system that, after the initial synthesis of the CoA ester, enables the acyl moiety to permeate membranes without the need to re-expend energy. In most cases, the cell achieves these requirements through a single mechanism, namely the reaction between CoA esters and L -carnitine to form the corresponding carnitine ester and regenerate unesterified CoA. The reversible reaction catalysed by a family of carnitine acyltransferases is shown in Fig. 1. The transfer to carnitine enables the cell to move the required moieties between intracellular compartments while keeping pools of CoA esters distinct in their respective compartments. The high impact that the carnitine acyltransferases have on the regulation of cellular metabolism derives to some extent from the limited availability of CoA in the intracellular compartments and also from the presence of effective mechanisms for

N H2

HIS

N

N O

H

O C

+

O

C

N

N H

CH 3

H

H

O

P

-

N

O

O

N

O-

C H3 OH

O

O

Substrates in the active site

CH3

N

O

P O

N H3C H3C

O H C H3

O

S

OH

-O

O

-O

P

O

O-

L-Carnitine

HIS

N H

CoA S

H

O

CH3

O C

-O

O-

+ N

Tetrahedral intermediate

H3C

CH3 CH3 H

O

CH3

O C

-O

+ O N

Products released

H3C

CH3 CH3

Fig. 1. The reaction of CrAT as proposed by Chase and Tubbs (1970). Evidence for the tetrahedral intermediate is reviewed in Colucci and Gandour (1988) and is supported by the crystal structure which gave the orientation of the reactive groups (Jogl and Tong, 2003). L -Carnitine is shown in black, CoA in green, and the acetate group in red. In the active site, the catalytic base is positioned so that it can pull a proton from either carnitine or CoA depending on the direction of the freely reversible reaction.

478

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

the transfer of carnitine and carnitine ester across different intracellular membrane systems. The high impact is also seen in the clinical manifestations of defects in the carnitine system, including seizures, heart failure and muscle weakness in mild cases and death for more serious defects.

2. The carnitine acyltransferases 2.1. The carnitine acyltransferases––locations The carnitine acyltransferases are a family of proteins that are widely distributed in the cell, and whose properties are specifically tailored to their complementary roles in overall involvement of carnitine in the maintenance of cell function (reviewed in van der Leij et al., 2000; Ramsay et al., 2001). The scheme in Fig. 2 illustrates the locations of the acyltransferases in the cell. There is only one transferase that has direct access to the cytosolic pool of acyl-CoA, the long-chain specific carnitine palmitoyltransferase 1 (CPT 1). Its most distinguishing kinetic characteristic is its

Fig. 2. Proteins of the carnitine system connect pools of acetyl-CoA. Malonyl-CoA sensitive enzymes such as CPT 1, shown as black squares, act on cytosolic long-chain substrates. CrAT is found only inside the organelles using matrix acetyl-CoA and acetyl-carnitine. The mitochondrial exchange carrier (CACT, striped squares) transfers carnitine and its esters across the membranes and similar transport proteins are suggested for the other organelles. The sodium-dependent organic cation transporter (OCTN2) is the high affinity carnitine transporter for uptake of carnitine into the cell but the mechanism for export from the cell is not well characterised.

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

479

binding of malonyl-CoA that regulates its activity. Another long-chain specific enzyme, CPT 2, is localised on the mitochondrial inner membrane with its catalytic site facing the matrix and a third in the peroxisomal matrix, carnitine octanoyltransferase (COT). The short acyl chain specific enzyme, carnitine acetyltransferase (CRAT) is localised within the mitochondrial matrix, in the peroxisomal core, in the endoplasmic reticular lumen, and has been reported in the nucleus, but is never found in the cytosol. The identity of the enzymes in the endoplasmic reticulum (ER) remains uncertain. Isolated ER has malonyl-CoA-sensitive CPT activity but immunoblotting failed to detect CPT 1. Recently, lumenal palmitoyl-CoA formation from added palmitoylcarnitine was demonstrated (Gooding et al., 2004). The synthesis of complex lipids (via, e.g., diacylglycerol acyltransferase and acyl cholesterol acyltransferase) inside the lumen of ER means that active acyl groups must be delivered there but the flux rate may be less critical than in mitochondria where the demands for fat breakdown and ketone body synthesis must be rapid and tightly regulated. The reversible removal or supply of activated acyl groups from or to the isolated CoA pools depends on the specific locations of the acyltransferases and acyl chain length specificity of the enzyme(s) in that location. For example, it is critical that, in contrast to yeast, there is no CrAT activity in the cytosol of mammalian cells (Abbas et al., 1998). The supply of acetyl-CoA required for fatty acid synthesis comes from citrate exported from the mitochondria and not from acetyl-carnitine. Acetyl-carnitine exported from the mitochondria is available for re-import so that an equilibrium between the acylation state of the carnitine pool in the cytosol and the CoA pool in the mitochondria is observed in liver (Bhuiyan et al., 1988). In sperm and in insect flight muscle, the cytosolic acetyl-carnitine serves as a readily available source of acetyl-CoA for energy production (reviewed in Bremer, 1983). For the long-chain acyl-carnitine pools, a similar buffering role was established by demonstrating the transfer of acyl groups from the carnitine pool into membrane phospholipids as part of the repair pathway (Arduini et al., 1992). The intracellular distribution of the acyltransferases is integrative and related to the requirement of every cell to regulate carbohydrate and lipid metabolism coordinately. CPT 1 (on the mitochondrial outer membrane) and CPT 2, which are both enriched in the mitochondrial contact sites (Fraser and Zammit, 1998), work in tandem to transfer long-chain acyl moieties from the cytosolic compartment to the mitochondrial matrix, where b-oxidation occurs (Fig. 2). Inhibition of CPT 1 by malonyl-CoA ensures that the flux through the two enzymes is closely regulated by the relative availability of glucose and fatty acids to the cell, as well as by hormonal (e.g., insulin, glucagon, leptin, adiponectin) and neuronal (e.g., sympathetic) inputs to the tissues. Therefore, the source of cytosolic malonyl-CoA to which CPT 1 is sensitive is of considerable importance. There is evidence that only a specific, possibly channelled, pool of malonyl-CoA is relevant in this respect. Malonyl-CoA is synthesised from acetyl-CoA through the catalytic activity of two isoforms of acetyl-CoA carboxylase: ACC-a and ACC-b. Whereas ACC-a appears to give rise to the bulk of the pool of malonyl-CoA within the cytosol that is used as a precursor for fatty acid synthesis,

480

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

ACC-b appears to supply a specific pool of malonyl-CoA that regulates the rate of mitochondrial fatty acid oxidation. This would be in accord with its localisation on the outer aspect of the mitochondrial outer membrane (the same as CPT 1). Thus, disruption of the ACC-b gene in mice results in increased rates of fatty acid oxidation not only in muscle (where this isoform predominates) but also in the liver, in which the overall content of malonyl-CoA does not change significantly, indicating that the absence of a small but compartmentalised pool of malonyl-CoA is involved in the regulation of L-CPT 1 (Abu-Elheiga et al., 2001). 2.2. The carnitine acyltransferases––properties A key requirement for the role of acyl-carnitine as a temporary storage form of activated acyl groups is the reversibility of the reaction catalysed by the carnitine acyltransferases. In all the isoenzymes studied, the equilibrium constant is close to 1. The reversible mechanism (Fig. 1) was elucidated by kinetic and inhibition studies and was validated by the recent structure of CrAT (Jogl and Tong, 2003). The active site of CrAT can orient the two substrates for reversible connection of the acyl group to either carnitine or CoA, facilitated by the catalytic base (HIS 343 in CrAT). Each member of the family has broad but different chain length specificity (reviewed in Ramsay et al., 2001). 2.3. The carnitine acyltransferases––deficiencies Since the first defects in fatty acid metabolism were characterised in the 1970s, many mutations that result in deficient activities have been reported (Kerner and Hoppel, 1998; Rebouche and Seim, 1998; Winter and Buist, 2000; Bartlett and Pourfarzam, 2002). Deficiencies in tissue carnitine, either primary or secondary, detract from the proper function of the carnitine system both by reducing the pool size and by decreasing the substrate level so that lower rates of transfer will be observed. Deficiencies in the intracellular carrier CACT will not only prevent delivery of activated fatty acids for oxidation but also will prevent the buffering of the mitochondrial acyl-CoA pool and thus influence glucose metabolism via pyruvate dehydrogenase (see later). CPT 2 deficiency is a common inborn error of mitochondrial fatty acid that shows considerable phenotypic variability. The adult, the infantile, and the perinatal forms all show autosomal recessive inheritance. The perinatal (always fatal) and infantile forms involve multiple organ systems. The adult CPT-II clinical phenotype is benign with episodes of myopathic symptoms triggered by, for example, high-intensity exercise. Metabolic characterisation of a patient with the common Ser113Leu mutation in the CPT 2 gene showed that the patient was normal glucose tolerant but severely insulin resistant. Carbohydrate oxidation was maximally increased, lipid oxidation was virtually absent, and neither changed during insulin stimulation (Haap et al., 2002). Since CPT 2 is induced by peroxisome proliferators (Brady et al., 1989), treatment of mild-type CPT 2-deficient human fibroblasts with the peroxisome proliferator,

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

481

bezafibrate, was tested as a way to stimulate fatty acid oxidation rates. The cells showed a time- and dose-dependent increase in CPT 2 mRNA and enzyme activity increased significantly leading to normalization of [3 H]-palmitate and [3 H]-myristate cellular oxidation rates (Djouadi et al., 2003). Carnitine palmitoyltransferase 1 (CPT 1) that catalyses the formation of acylcarnitine, the first step in the oxidation of long-chain fatty acids in mitochondria, exists as liver (L-CPT 1) and muscle (M-CPT 1) isoforms that are encoded by separate genes (reviewed in van der Leij et al., 2000). Genetic deficiency of L-CPT I is characterized by episodes of hypoketotic hypoglycemia beginning in early childhood and usually associated with fasting or illness. The mutations underlying L-CPT I deficiency can now be analysed by heterologous expression, as for example G709E and G710E which abolish activity (Gobin et al., 2003). M-CPT 1 deficiency is still unknown.

3. The carnitine system protects the free CoA pools 3.1. Evidence for acyl interchange between carnitine and CoA pools in the cell and animal Experiments on isolated mitochondria from rat heart and liver compared the acylation (acetyl and succinyl) state of the mitochondrial CoA pool in state 3 and state 4 respiration and the effect of adding carnitine to the incubation (Lysiak et al., 1988). The total CoA in heart mitochondria was 1.6–2.0 nmol/mg but was 2.4–2.8 nmol/mg mitochondrial protein in liver mitochondria and the CoA pool was more acylated in state 4 than in state 3 (80% versus 67% in heart and 55% versus 50% in liver). Added carnitine decreased the ratio of acetyl-CoA/free CoA by 10 fold in heart but less than 2-fold in liver with either pyruvate or octanoate as substrate. When the acetyl-CoA was less than 10% of the pool, it was preferentially used for the TCA cycle rather than exported as acetyl-carnitine but above that the export of acetyl-carnitine increased linearly with the acylation ratio. The mitochondrial pool of CoA ranges from 95% of the cellular CoA in heart (Idell-Wenger et al., 1978) to about 44% in liver (Brass and Ruff, 1992), giving a matrix concentration of the order of 2 mM (Lysiak et al., 1988). However, the cytosolic concentration of CoA may be as low as 5 nM even in liver, a concentration inferred by the lack of inhibition of acetyl-CoA carboxylase that has a Ki for acetylCoA of 5 nM (Faergeman and Knudsen, 1997). In liver, the carnitine content per gram of liver is about 3.7 times higher than the CoA content (Bhuiyan et al., 1988) and is mainly non-mitochondrial (90%) in heart (Idell-Wenger et al., 1978). The acylation state of the cytosolic pool of carnitine reflects the acylation state of the mitochondrial CoA indicating that the reaction catalysed by CrAT is near equilibrium in vivo (Bhuiyan et al., 1988). Two reports suggest that the cytosolic pool is very rapidly reflected in the serum carnitine acylation state. Injection of carnitine into rats resulted in a large efflux of acyl-carnitines into serum within 5 min (Brass and Hoppel, 1980). The second report

482

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

noted a decrease in serum acetyl-carnitine within 5 min of refeeding glucose to starved mice (Yamaguti et al., 1996). Thus, this is a practical export mechanism for excess acyl groups, a phenomenon also seen in the relief of effects of incipient ischemia in rat Langerdoff hearts where export of acyl-carnitine is accompanied by less appearance of lactate (Hulsmann et al., 1996). The acylation state of the serum carnitine pool varies with metabolic state. The acetylation of the carnitine pool increases from 37% in fed rats to 53% in starved rats where the level increases further to 73% after carnitine supplementation (Brass and Hoppel, 1980). In mice, a similar increase in acetyl-carnitine was observed during fasting but it was rapidly reversed by glucose feeding when the acetyl groups appeared in the liver (Yamaguti et al., 1996). Accompanied by the rapid uptake of acetyl-carnitine into monkey brain (Kuratsune et al., 1997) (see below), these observations suggest that acetyl-carnitine can also serve as an alternative fuel for brain during starvation. 3.2. Carnitine in metabolic therapy The equilibrium of the acylation state of the limited CoA pools with the large cellular pool of carnitine is the basis for the use of carnitine as a therapy in conditions where abnormal, excess, or non-metabolised organic acid esters accumulate in the cell. From the mitochondria, where free CoA is critical for the TCA cycle and all aerobic energy generation, acyl-carnitine is transported via CACT to the cytosol where the larger pool of carnitine (about 90% of the cell content) dilutes the accumulation. From the cytosol, acyl-carnitine is readily exported by a still unidentified mechanism (Sandor et al., 1985) down the concentration gradient from 0.5–1 mM in the cell to 50 lM total carnitine in the plasma. The acylation state of the plasma carnitine pool reflects the accumulation of excess intracellular acyl groups as illustrated by the diagnostic profiles of acyl-carnitine derivatives in patients with defects of boxidation. Analysis of the acyl-carnitine profile in urine or blood spots is now standard practice for diagnosis (Fingerhut et al., 2001; Sim et al., 2001; Gempel et al., 2002). Diabetic patients excrete more long-chain carnitine esters (C12–C16) than controls so that it was suggested that the urinary acyl-carnitine pattern determined by electrospray ionization-mass spectrometry might be a useful tool for diagnosis of and monitoring therapy in diabetes mellitus (Moder et al., 2003). In the kidney, carnitine and all esters are excreted but only carnitine and its short-chain esters are efficiently reabsorbed (98–99%) (Evans and Fornasini, 2003) up to their level in plasma carnitine. Thus, excess acyl groups can be exported so long as there is sufficient carnitine in the plasma. Appearance of acyl-carnitine in the urine of diseased patients can amount to a gram per day and so carnitine supplementation (usually 10–50 mg/kg/day) is critical to the continued flow. In 1992, carnitine was approved by the United States Food and Drug Administration for the treatment of inborn errors of metabolism where abnormal acyl-CoA derivatives accumulate because the excretion increased with carnitine supplementation and correlated with improved clinical status. For example, patients with 2-

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

483

methylacetoacetyl-CoA thiolase deficiency showed increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Excessive amounts of short and branched chain acyl-carnitines were excreted in urine and these increased in response to L -carnitine (Fontaine et al., 1996). Similar beneficial effect is found where long-chain acyl groups accumulate, such as in very long-chain acylCoA dehydrogenase (VLCAD) deficiency. In both control and VLCAD knockout mice, an increase in blood acyl-carnitines (C14–C18) (and presumably elevated excretion) with the concomitant decrease in free carnitine was observed in fasted mice. In VLCAD knockout mice, the exaggerated response to an 8 h fast with the added stress of cold exposure resulted in a five-fold increase in long-chain acylcarnitines and four out of the 12 knockout mice died (Spiekerkoetter et al., 2004). Although cellular acylation state was not studied, the flux of long-chain acyl carnitines into the blood is indicative of increased long-chain acylation of the CoA pools and the 33% fatal outcome suggests that this compromises cell and hence organ function. Where drug metabolism produces organic acids, removal of the breakdown products is critical and is facilitated by carnitine (reviewed in Arrigoni-Martelli and Caso, 2001). In the case of the antiepileptic drug, valproate, Reyes syndrome (associated with overacylation of the mitochondrial CoA pool) could develop but can be prevented by carnitine supplementation. Another major class of drugs where excess acylated carnitine is found in urine is the pivaloyl antibiotics (Brass, 1994). Excretion as pivaloyl-carnitine is the only mechanism of detoxification and can result in plasma and muscle carnitine levels as low as 10% of normal. Secondary carnitine deficiency can develop after these drug therapies and also in patients on long-term dialysis, resulting in high fasting levels of ketones, high acylcarnitine/free carnitine ratios in blood, and high plasma and liver lipid levels. Carnitine supplementation reversed the effects. It has been suggested (Steiber et al., 2004) that the need for carnitine supplementation in dialysis patients is more critical in patients with existing cardiac hypertrophy and in patients on aspirin therapy (salicylic-CoA inhibits CPT 2, Vessey et al., 1991). Pediatric cancer patients have also been reported to have secondary carnitine deficiency not due to inadequate nutrition levels (Yaris et al., 2002). Metabolic changes that result from therapy and/ or from neoplastic processes may be responsible for the decrease in carnitine levels. Carnitine is also a popular supplement in sports medicine where it is believed to improve muscle performance especially in endurance sports. The influence of the carnitine system in balancing cellular fuel use (see below) may be part of the mechanism. However, convincing studies for its efficacy without prior carnitine deficiency are lacking (Heinonen, 1996; Maughan et al., 2004). 3.3. Therapeutic use of short-chain acyl-carnitine esters Long-chain acyl carnitine esters are excreted after a heart attack, so carnitine was suggested as a means of improving clearance of the damaging esters. However, propionyl-L -carnitine proved much more therapeutically beneficial, possibly because propionate replenishes mitochondria with intermediates of the citric acid cycle to

484

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

stimulate energy production in the post-ischaemic reperfusion phase. The increased cellular content of carnitine may contribute to normalisation of the glucose/fat fuel balance by its effect on the acetyl-CoA/CoA ratio in mitochondria. In a study in diabetic rats, oral administration of propionyl-L -carnitine reduced abnormalities of cardiac function and decreased plasma lipids (Terada et al., 1998). Post-ischaemic diabetic rat heart function was improved following chronic propionyl-L -carnitine treatment and mitochondrial deficits in pyruvate oxidation were reversed (Felix et al., 2001). Improved energy status with carnitine ester therapy was demonstrated by NMR spectroscopy of the high energy phosphates (Loster et al., 1999). The therapeutic use of acetyl-L -carnitine has opened a fascinating window on the role of acetyl-CoA in brain function. Aureli et al. (1998, 2000) observed that acetylcarnitine treatment increased glycogen synthesis in the brain and that long-term acetyl-carnitine treatment normalised age-related disturbances in brain function. Acetyl-L -carnitine significantly reversed age-associated decline in mitochondrial membrane potential and cardiolipin content, and improved ambulatory activity (Hagen et al., 1998). Feeding rats acetyl-carnitine with lipoic acid, an antioxidant, increased metabolism, restored mitochondrial function and lowered oxidative stress in old rats (Hagen et al., 2002) and also increased their ambulatory activity and cognition (Liu et al., 2002). Improvement of memory deficits in Alzheimer’s disease has been noted in several studies (Ando et al., 2001; Arrigoni-Martelli and Caso, 2001; Montgomery et al., 2003). Acetyl-L -carnitine (ALCAR) has recently been reported to improve MS-related fatigue (Tomassini et al., 2004). In an open randomised trial of 2 g/day for 24 weeks, acetyl-carnitine significantly improved mental fatigue and propionyl-carnitine improved general fatigue (Vermeulen and Scholte, 2004). In the acetyl-carnitine group only, the changes in plasma carnitine levels correlated with clinical improvement. A Positron Emission Tomography (PET) study of acetate incorporation from ALCAR suggested that levels of biosynthesis of neurotransmitters from acetyl-carnitine might be reduced in some brain regions of chronic fatigue patients and that this abnormality might be one of the keys to unveiling the mechanisms of the chronic fatigue sensation (Kuratsune et al., 2002). A PET study in monkeys demonstrated that the carnitine uptake into brain was slow but when the ALCAR was labelled in on the carbon 2 of acetate, accumulation was higher. Thus there was net accumulation of the acetate part of ALCAR but not its carnitine part indicating rapid metabolism of the acetate moiety (Kuratsune et al., 1997). ALCAR accumulation was deficient in jvs mice (Kido et al., 2001). The question is where that acetate goes. It could be used for energy, for incorporation into lipids or proteins or, as recently suggested, to increase the production of glutamate. In a study of glucose analog uptake in brain slices, results indicated that acetyl-carnitine might be used for the production of releasable glutamate rather than as an energy source (Tanaka et al., 2003). Another study found that carnitine supplementation increased the levels of dopamine, epinephrine, and serotonin particularly in regions rich in cholinergic neurons (Juliet et al., 2003). In a careful NMR study, the [13 C] label from [(1,2-[13 C](2))acetyl]-L -carnitine was also found in liver glutamate, glutamine, and glutathione (Aureli et al., 1999) in accord with entry into the mitochondrial acetyl-CoA pool associated with the tricarboxylic acid cycle. In

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

485

brain, after injection of [1-14 C]acetyl-L -carnitine, 60% of the radioactivity was recovered as CO2 consistent with energy generation. The percentage of radioactivity recovered in brain after 6 h was about 1%, 25% of which was in polyunsaturated fatty acids incorporated into complex lipids (Ricciolini et al., 1998). In contrast, glucose did not contribute acetate to the polyunsaturated fatty acid pool at all. The beneficial effects of ALCAR in brain could come from any or all of these pathways.

4. The influence of the carnitine system on intracellular metabolism 4.1. Fuel use Using heart as an example, we now consider the intracellular processes of metabolic integration and fuel use that underlie the therapeutic benefits of (acyl)carnitine supplementation. In cardiac ischemia there is a relative deficit of oxygen availability. One strategy for improving outcomes is to optimise cardiac function in relation to oxygen availability (Lopaschuk, 2004) by improving the balance between fatty acid and pyruvate (glucose) utilisation by mitochondria. Increased fatty acid oxidation feeds back to inhibit glucose oxidation at several steps (glucose uptake, phosphorylation and pyruvate dehydrogenase activity). Even if glucose is processed through glycolysis, oxidation of pyruvate is inhibited, increasing the production of lactate and lowering intracellular pH, thus altering trans-membrane movements of Ca2þ and Naþ which increase the ATP requirement of the tissue at a time when formation of ATP is diminished (Dennis et al., 1991; Liu et al., 2002). As fatty acid oxidation recovers more quickly during cardiac ischaemia, glycolytic formation of lactate is increased, with a consequent decline in cardiac efficiency. Carnitine and propionyl-L -carnitine are beneficial during ischaemia (Retter, 1999). The effects of ischaemia and post-ischaemia are exacerbated by the continued high utilisation of fatty acids by the myocardium (Stanley et al., 1997; Lopaschuk et al., 1999). Carnitine has been shown to inhibit fatty acid oxidation by the myocardium, and may thus have a beneficial effect under these conditions. The inhibition may appear paradoxical as carnitine is a substrate for CPT1, but as shown in Fig. 3 the increased availability of carnitine inside the mitochondria will lower acetyl-CoA levels there, through the action of CrAT, thus de-inhibiting pyruvate dehydrogenase and increasing the oxidation of pyruvate (while lowering the rate of formation of lactate and normalising pH, Zammit, 1998). Propionyl-L -carnitine would also be predicted to have the same effect, but with the added benefit of generating ATP through the metabolism of the propionyl moiety (Wiseman and Brogden, 1998; Zammit, 1998). This has been shown experimentally in hypertrophied hearts (Schonekess et al., 1995) and post-ischaemically (Loster et al., 1999; Felix et al., 2001). However, not all studies have shown a universal benefit for cardiac function (Ferrari and De Giuli, 1997). As predicted from Fig. 3, acetyl-carnitine was shown not to be so effective in these studies. Propionyl-L -carnitine has also been shown to be a good superoxide anion scavenger (Vanella et al., 2000).

486

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493 glucose LC-CoA

carnitine CPT 1 pyruvate cytosol

Mitochondrial matrix

LC-carnitine pyruvate

PDH

-

acetylcarnitine

acetyl-CoA

CRAT acetyl-CoA

TCA cycle

Fig. 3. Anticipated effects of increased L -carnitine availability on cardiac glucose-fatty acid interactions. The mass-action effect of carnitine on CPT 1 activity is counteracted by that on CrAT, which lowers intramitochondrial acetyl-CoA, and thus de-inhibits PDH, to allow higher pyruvate oxidation rates.

4.2. Carnitine-mediated peroxisomal–mitochondrial interactions It has long been known that there is a route for partially oxidised products of peroxisomal fatty acid oxidation to be further processed by mitochondria. Peroxisomes partly oxidise very-long-chain fatty acids, generating shorter ones for transport to the mitochondria for complete oxidation, although shortened acyl chains are also substrates for microsomal processes such as desaturation and subsequent reelongation. The presence of COT within peroxisomes is assumed to provide the mechanism whereby medium-chain acyl-CoA esters are transesterified to acyl-carnitines for transport elsewhere. COT can only function in this capacity if it has wide chain-length specificity, as indeed it does, with species differences in the optimum chain length (Ramsay, 1999). Acetyl-CoA is also a product of peroxisomal fatty acid oxidation and, in liver, peroxisomes deliver acetyl units to mitochondria both as acetate and acetyl-carnitine (Tran and Christophersen, 2001). A study published recently increases the possibilities for mitochondrial–peroxisomal interactions (Reszko et al., 2004). The findings indicated that the source of acetyl-CoA for malonyl-CoA synthesis in rat heart could also be less straightforward than hitherto realised. They suggest that, whereas most of the malonyl-CoA derived from pyruvate originates from mitochondrial acetyl-CoA (via the sequential catalytic actions of citrate synthase and ATP-citrate lyase) that derived from fatty acids (not necessarily very-long-chain ones) originates primarily within the peroxisomes.

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

487

Peroxisomes β -oxidation

acetylcarnitine

VLC-CoA

CRAT acetyl-CoA

acetate

LC-CoA

?

acetate acetyl-CoA

malonyl-CoA

-

CPT 1

LC-carnitine

acetylcarnitine

CRAT acetyl-CoA

β -oxidation

LC-CoA

Mitochondria Fig. 4. Possible routes for the transfer of acetyl units from peroxisomes to the mitochondria and the role that peroxisomal fatty acid oxidation may play as a source of cytosolic malonyl-CoA. b-Oxidation in the peroxisomal core results in the formation of acetyl-CoA, and, through CrAT activity, of acetyl-carnitine. Deacylation also generates acetate, at least in the liver. The absence of CrAT from the cytosol enables acetyl-carnitine (which may be the major product leaving the peroxisomes) to carry acetyl moieties to the mitochondria, for oxidation through the TCA cycle. Smaller amounts of acetyl-CoA and acetate reaching the cytosol may be sufficient to modulate malonyl-CoA concentrations and regulate CPT 1 activity.

This suggests that acetyl-CoA generated from incomplete b-oxidation within peroxisomes gives rise to cytosolic acetyl-CoA without equilibration with the mitochondrial acetyl-CoA pool, at least in heart (Fig. 4). This implies that peroxisomal fatty acid oxidation participates in the control of mitochondrial fatty acid oxidation (Fig. 4). It is instructive to analyse the possible mechanisms that could lead to acetyl group exchange between the peroxisomal and cytosolic compartments, in view of the fact that CrAT is present in peroxisomes but absent from the cytosol. It has long been assumed that the presence of CrAT in peroxisomes enables the traffic of acetylcarnitine formed from the peroxisomal oxidation product, acetyl-CoA, to the mitochondria for oxidation. However, these recent data appear to indicate that the process can be interrupted in the cytosol even in the absence of any obvious means of re-generating acetyl-CoA in the cytosol from acetyl-carnitine. This implies that acetyl-CoA destined for malonyl-CoA synthesis exits from the peroxisomes as

488

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

acetyl-CoA. As mentioned in Section 1, the peroxisomal membrane appears to be an exception in that acyl-CoA esters are able to be trafficked across it, so the exit of acetyl-CoA directly, without the preliminary formation of acetyl-carnitine may be feasible, but further studies are required to ascertain whether this can occur. This ability, coupled with the absence of CrAT from the cytosol may be a mechanism for diverting acetyl moieties partly to mitochondria (as acetyl-carnitine) and partly to the cytosol (as acetyl-CoA). In the liver, the situation is complicated further by the ability of peroxisomes to release the product of oxidation partly as acetate, although this should be readily re-esterified to acetyl-CoA through the presence of cytosolic acetyl-CoA synthase in this tissue. 4.3. Intracellular effects of therapeutic use of carnitine and its esters The intracellular distribution of the various carnitine acyltransferases may also be important in the roles that carnitine itself (Fig. 3) and short-chain acyl-carnitines (Figs. 5 and 6) appear to play as pharmacological agents in the alleviation of conditions as diverse as dementia and cardiac ischaemic stress. It has been known for some time that carnitine, and its short-chain esters (acetyl-carnitine and especially propionyl-carnitine) are able to improve metabolic function, as discussed above. The

Peroxisomes

acetylcarnitine

acetylcarnitine

CRAT ? acetyl-CoA

acetyl-CoA

LC-CoA

ACC malonyl-CoA

-

acetate

CPT 1

pyruvate

Mitochondrial matrix acetylcarnitine

LC-carnitine

CPT 2

pyruvate

CRAT

PDH acetyl-CoA

LC-CoA

TCA cycle

Fig. 5. Acetyl-carnitine may be used as an ATP-generating substrate in peripheral tissues, while inhibiting the utilisation of fatty acids and glucose, which are alternative substrates. The organellar distribution of the various carnitine acyltransferases is critical for the interactions that enable the glucose- and fatty acidsparing effects of acetyl-carnitine.

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

489

Peroxisome

LC-CoA malonyl-CoA

-

glucose

acetyl-CoA

CPT 1

CRAT propionylcarnitine

acetylcarnitine

pyruvate

cytosol Mitochondrial matrix

acetylcarnitine

propionylcarnitine LC-carnitine

CPT 2

PDH

CRAT CRAT

carnitine

acetyl-CoA

-

LC-CoA propionyl-CoA

oxaloacetate

ATP

β -oxidation

TCA cycle

Fig. 6. Propionyl-L -carnitine metabolism favours pyruvate (glucose) oxidation but antagonises that of fatty acids. Increased acetyl-carnitine availability may generate increased cytosolic concentrations of malonyl-CoA (through interactions with peroxisomes) and lower intramitochondrial acetyl-CoA. The metabolism of the propionyl moiety through the second half of the TCA cycle generates reducing equivalents that are used for GTP and ATP formation, as well as generating oxaloacetate that, through reaction with acetyl-CoA to generate citrate, will further lower intramitochondrial acetyl-CoA and generate cytosolic malonyl-CoA. Thus, both parts of the propionyl-L -carnitine molecule act in synchrony to alter the balance between glucose and fatty acid utilisation in tissues (e.g. in heart).

superiority of propionyl-carnitine in this respect may be related to the anticipated metabolic effects of the carnitine and propionyl entities as compared to those of carnitine by itself or of the acetyl moiety in acetyl-carnitine. Thus, propionyl-carnitine is expected to give rise to propionyl-CoA and carnitine through the action of intramitochondrial CrAT (Fig. 6). The propionyl-CoA will feed into the second half of the TCA cycle generating ATP and oxaloacetate in the process, while carnitine will equilibrate with acetyl-CoA (again through CrAT activity). Therefore, both the carnitine and propionyl moieties will tend to lower the intra-mitochondrial content of acetyl-CoA which will result in the de-inhibition of pyruvate dehydrogenase and increased oxidation of pyruvate. This is associated with increased cardiac efficiency especially when accompanied by decreased fatty acid oxidation. The increased formation of acetyl-carnitine may additionally help to inhibit mitochondrial fatty acid oxidation too, if it is able to generate acetyl-CoA within the peroxisomes (through a reversal of the process depicted in Fig. 4) and thereby cytosolic acetyl-CoA as a substrate for malonyl-CoA formation (Fig. 6). It can be seen from the above that the localisation of CrAT (which is targeted to peroxisomes and mitochondria simultaneously by virtue of the production of the relevant targeting motifs through alternative splicing) may play a significant role in mitochondrial–peroxisomal metabolic interactions that extend beyond the ability of

490

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

mitochondria to process the products of peroxisomal fatty acid oxidation. Although no experimental evidence exists, it is possible that the rapid formation of acetylcarnitine as a result of a high rate of fatty acid oxidation within mitochondria could, through this peroxisomal cycling, raise cytosolic malonyl-CoA levels so as to provide feedback inhibition, and thus restraint, of the mitochondrial fatty acid oxidation. 4.4. Concluding remarks Carnitine clearly provides a means to increase export of excess acyl groups from cells and, in particular, the mitochondrial pool of CoA that is essential to aerobic metabolism. The exact mechanism of the influence on fuel use and metabolic regulation still need further work. The effect on pyruvate dehydrogenase is relatively well delineated but the source of acetate for the production of malonyl-CoA that regulates CPT 1 as opposed to providing acetate for fatty acid synthesis is an interesting new development. Understanding the normal mechanisms for the effects of the carnitine system in buffering the CoA pools is still unfolding, and its beneficial effects in clinical use depend on the changes induced in these pools.

References Abbas, A.S., Wu, G.X., Schulz, H., 1998. Carnitine acetyltransferase is not a cytosolic enzyme in rat heart and therefore cannot function in the energy-linked regulation of cardiac fatty acid oxidation. J. Mol. Cell. Cardiol. 30, 1305–1309. Abu-Elheiga, L., Matzuk, M.M., Abo-Hashema, K.A.H., Wakil, S.J., 2001. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science 291, 2613–2616. Ando, S., Tadenuma, T., Tanaka, Y., Fukui, F., Kobayashi, S., Ohashi, Y., Kawabata, T., 2001. Enhancement of learning capacity and cholinergic synaptic function by carnitine in aging rats. J. Neurosci. Res. 66, 266–271. Arduini, A., Mancinelli, G., Radatti, G.L., Dottori, S., Molajoni, F., Ramsay, R.R., 1992. Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty-acid turnover in intact human erythrocytes. J. Biol. Chem. 267, 12673–12681. Arrigoni-Martelli, E., Caso, V., 2001. Carnitine protects mitochondria and removes toxic acyls from xenobiotics. Drug Exp. Clin. Res. 27, 27–49. Aureli, T., Di Cocco, M.E., Pucetti, C., Ricciolini, R., Scalibastri, M., Miccheli, A., Manetti, C., Conti, F., 1998. Acetyl-L -carnitine modulates glucose metabolism and stimulates glycogen synthesis in rat brain. Brain Res. 796, 75–81. Aureli, T., Puccetti, C., Di Cocco, M.E., Arduini, A., Ricciolini, R., Scalibastri, M., Manetti, C., Conti, F., 1999. Entry of (1,2-C-13(2))acetyl-L -carnitine in liver tricarboxylic acid cycle and lipogenesis––A study by C-13 NMR spectroscopy in conscious, freely moving rats. Eur. J. Biochem. 263, 287–293. Aureli, T., Di Cocco, M.E., Capuani, G., Ricciolini, R., Manetti, C., Miccheli, A., Conti, F., 2000. Effect of long-term feeding with acetyl-L -carnitine on the age-related changes in rat brain lipid composition: A study by P-31 NMR spectroscopy. Neurochem. Res. 25, 395–399. Bartlett, K., Pourfarzam, M., 2002. Defects of beta-oxidation including carnitine deficiency. Mitochondrial Function Dysfunction 53, 469–516. Bhuiyan, A., Bartlett, K., Sherratt, H.S.A., Agius, L., 1988. Effects of Ciprofibrate and 2-5-(4chlorophenyl)pentyl oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme-activities in rats––relation between hepatic carnitine concentration and carnitine acetyltransferase activity. Biochem. J. 253, 337–343.

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

491

Brady, P.S., Marine, K.A., Brady, L.J., Ramsay, R.R., 1989. Coordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs. Biochem. J. 260, 93–100. Brass, E.P., 1994. Overview of coenzyme––a metabolism and its role in cellular toxicity. Chem.-Biol. Interact. 90, 203–214. Brass, E., Hoppel, C.L., 1980. Relationship between acid-soluble carnitine and CoA pools in vivo. Biochem. J. 190, 495–504. Brass, E.P., Ruff, L.J., 1992. Rat hepatic Coenzyme A is redistributed in response to mitochondrial acylcoenzyme A accumulation. J. Nutr. 122, 2094–2100. Bremer, J., 1983. Carnitine––metabolism and functions. Physiol. Rev. 63, 1420–1480. Chase, J.F.A., Tubbs, P.K., 1970. Biochem. J. 116, 713–720. Colucci, W.J., Gandour, R.D., 1988. Carnitine acetyltransferase––a review of its biology, enzymology, and bioorganic chemistry. Bioorg. Chem. 16, 307–334. Dennis, S.C., Gevers, W., Opie, L.H., 1991. Protons in ischemia––where do they come from––where do they go to. J. Mol. Cell. Cardiol. 23, 1077–1086. Djouadi, F., Bonnefont, J.P., Thuillier, L., Droin, V., Khadom, N., Munnich, A., Bastin, J., 2003. Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate. Pediatr. Res. 54, 446–451. Evans, A.M., Fornasini, G., 2003. Pharmacokinetics of L -carnitine. Clin. Pharmacokinet. 42, 941–967. Faergeman, N.J., Knudsen, J., 1997. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem. J. 323, 1–12. Felix, C., Gillis, M., Driedzic, W.R., Paulson, D.J., Broderick, T.L., 2001. Effects of propionyl-L -carnitine on isolated mitochondrial function in the reperfused diabetic rat heart. Diabetes Res. Clin. Pract. 53, 17–24. Ferrari, R., De Giuli, F., 1997. The propionyl-L -carnitine hypothesis: an alternative approach to treating heart failure. J. Card. Fail. 3, 217–224. Fingerhut, R., Roschinger, W., Muntau, A.C., Dame, T., Kreischer, J., Arnecke, R., Superti-Furga, A., Troxler, H., Liebl, B., Olgemoller, B., Roscher, A.A., 2001. Hepatic carnitine palmitoyltransferase I deficiency: acylcarnitine profiles in blood spots are highly specific. Clin. Chem. 47, 1763–1768. Fontaine, M., Briand, G., Ser, N., Armelin, I., Rolland, M.O., Degand, P., Vamecq, J., 1996. Metabolic studies in twin brothers with 2-methylacetoacetyl-CoA thiolase deficiency. Clin. Chim. Acta 255, 67– 83. Fraser, F., Zammit, V.A., 1998. Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria. Biochem. J. 329, 225–229. Gempel, K., Kiechl, S., Hofmann, S., Lochmuller, H., Kiechl-Kohlendorfer, U., Willeit, J., Sperl, W., Rettinger, A., Bieger, I., Pongratz, D., Gerbitz, K.D., Bauer, M.F., 2002. Screening for carnitine palmitoyltransferase II deficiency by tandem mass spectrometry. J. Inherit. Metab. Dis. 25, 17–27. Gobin, S., Thuillier, L., Jogl, G., Faye, A., Tong, L., Chi, M., Bonnefont, J.P., Girard, J., Prip-Buus, C., 2003. Functional and structural basis of carnitine palmitoyltransferase 1A deficiency. J. Biol. Chem. 278, 50428–50434. Gooding, J.M., Shayeghi, M., Saggerson, E.D., 2004. Membrane transport of fatty acylcarnitine and free L -carnitine by rat liver microsomes. Eur. J. Biochem. 271, 954–961. Haap, M., Thamer, C., Machann, J., Tschritter, O., Loblein, K., Kellerer, M., Schick, F., Jacob, S., Haring, H.U., Stumvoll, M., 2002. Metabolic characterization of a woman homozygous for the Ser113Leu missense mutation in carnitine palmitoyl transferase II. J. Clin. Endocrinol. Metab. 87, 2139–2143. Hagen, T.M., Ingersoll, R.T., Wehr, C.M., Lykkesfeldt, J., Vinarsky, V., Bartholomew, J.C., Song, M.H., Ames, B.N., 1998. Acetyl-L -carnitine fed to old rats partially restores mitochondrial function and ambulatory activity. Proc. Natl. Acad. Sci. USA 95, 9562–9566. Hagen, T.M., Liu, J.K., Lykkesfeldt, J., Wehr, C.M., Ingersoll, R.T., Vinarsky, V., Bartholomew, J.C., Ames, B.N., 2002. Feeding acetyl-L -carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc. Natl. Acad. Sci. USA 99, 1870–1875. Heinonen, O.J., 1996. Carnitine and physical exercise. Sports Med. 22, 109–132.

492

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

Hettema, E.H., Tabak, H.F., 2000. Transport of fatty acids and metabolites across the peroxisomal membrane. BBA-Mol. Cell. Biol. Lipids 1486, 18–27. Hulsmann, W.C., Peschechera, A., Serafini, F., Ferrari, L.E., 1996. Release of ischemia in paced rat Langendorff hearts by supply of L -carnitine: Role of endogenous long-chain acylcarnitine. Mol. Cell. Biochem. 156, 87–91. Idell-Wenger, J., Grotyohann, L., Neely, J., 1978. CoA and carnitine distribution in normal and ischemic hearts. J. Biol. Chem. 253, 4310–4318. Jogl, G., Tong, L., 2003. Crystal structure of carnitine acetyltransferase and implications for the catalytic mechanism and fatty acid transport. Cell 112, 113–122. Juliet, P.A.R., Balasubramaniam, D., Balasubramaniam, N., Panneerselvam, C., 2003. Carnitine: a neuromodulator in aged rats. J. Gerontol. Ser. A-Biol. Sci. Med. Sci. 58, 970–974. Kerner, J., Hoppel, C., 1998. Genetic disorders of carnitine metabolism and their nutritional management. Annu. Rev. Nutr. 18, 179–206. Kido, Y., Tamai, I., Ohnari, A., Sai, Y., Kagami, T., Nezu, J., Nikaido, H., Hashimoto, N., Asano, M., Tsuji, A., 2001. Functional relevance of carnitine transporter OCTN2 to brain distribution of L -carnitine and acetyl-L -carnitine across the blood-brain barrier. J. Neurochem. 79, 959–969. Kuratsune, H., Watanabe, Y., Yamaguti, K., Jacobsson, G., Takahashi, M., Machii, T., Onoe, H., Onoe, K., Matsumura, K., Valind, S., Kitani, T., Langstrom, B., 1997. High uptake of 2-(11) acetyl-L carnitine into the brain: a pet study. Biochem. Biophys. Res. Commun. 231, 488–493. Kuratsune, H., Yamaguti, K., Lindh, G., Evengard, B., Hagberg, G., Matsumura, K., Iwase, M., Onoe, H., Takahashi, M., Machii, T., Kanakura, Y., Kitani, T., Langstrom, B., Watanabe, Y., 2002. Brain regions involved in fatigue sensation: reduced acetylcarnitine uptake into the brain. Neuroimage 17, 1256–1265. Liu, J.K., Killilea, D.W., Ames, B.N., 2002. Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetylL -carnitine and/or R-alpha-lipoic acid. Proc. Natl. Acad. Sci. USA 99, 1876–1881. Lopaschuk, G.D., 2004. Targets for modulation of fatty acid oxidation in the heart. Curr. Opin. Investig. Drugs 5, 290–294. Lopaschuk, G.D., Kantor, P.F., Dyck, J.R.B., 1999. Optimising cardiac metabolism: A new reference approach to the management of ischemic heart disease? Medicographia 21, 109–115. Loster, H., Keller, T., Grommisch, J., Grunder, W., 1999. Effects of L -carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of P-31-NMR spectroscopy. Mol. Cell. Biochem. 200, 93–102. Lysiak, W., Lilly, K., Dilisa, F., Toth, P.P., Bieber, L.L., 1988. Quantitation of the effect of L -carnitine on the levels of acid-soluble short-chain acyl-coa and coash in rat-heart and liver-mitochondria. J. Biol. Chem. 263, 1151–1156. Maughan, R.J., King, D.S., Lea, T., 2004. Dietary supplements. J. Sports Sci. 22, 95–113. Moder, M., Kiessling, A., Loster, H., Bruggemann, L., 2003. The pattern of urinary acylcarnitines determined by electrospray mass spectrometry: a new tool in the diagnosis of diabetes mellitus. Anal. Bioanal. Chem. 375, 200–210. Montgomery, S.A., Thal, L.J., Amrein, R., 2003. Meta-analysis of double blind randomized controlled clinical trials of acetyl-L -carnitine versus placebo in the treatment of mild cognitive impairment and mild Alzheimer’s disease. Int. Clin. Psychopharmacol. 18, 61–71. Ramsay, R.R., 1999. The role of the carnitine system in peroxisomal fatty acid oxidation. Am. J. Med. Sci. 318, 28–35. Ramsay, R.R., Gandour, R.D., van der Leij, F.R., 2001. Molecular enzymology of carnitine transfer and transport. Biochim. Biophys. Acta-Protein Struct. Molec. Enzym. 1546, 21–43. Rebouche, C.J., Seim, H., 1998. Carnitine metabolism and its regulation in microorganisms and mammals. Annu. Rev. Nutr. 18, 39–61. Reszko, A.E., Kasumov, T., David, F., Jobbins, K.A., Thomas, K.R., Hoppel, C.L., Brunengraber, H., Des Rosiers, C., 2004. Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-coa in rat heart. J. Biol. Chem. 279, 19574–19579. Retter, A.S., 1999. Carnitine and its role in cardiovascular disease. Heart Dis. 1, 108–113.

R.R. Ramsay, V.A. Zammit / Molecular Aspects of Medicine 25 (2004) 475–493

493

Ricciolini, R., Scalibastri, M., Kelleher, J.K., Carminati, P., Calvani, M., Arduini, A., 1998. Role of acetyl-L -carnitine in rat brain lipogenesis: implications for polyunsaturated fatty acid biosynthesis. J. Neurochem. 71, 2510–2517. Sandor, A., Kispal, G., Melegh, B., Alkonyi, I., 1985. Release of carnitine from the perfused rat-liver. Biochim. Biophys. Acta 835, 83–91. Schonekess, B.O., Allard, M.F., Lopaschuk, G.D., 1995. Propionyl L -carnitine improvement of hypertrophied heart function is accompanied by an increase in carbohydrate oxidation. Circ. Res. 77, 726–734. Sim, K.G., Wiley, V., Carpenter, K., Wilcken, B., 2001. Carnitine palmitoyltransferase I deficiency in neonate identified by dried blood spot free carnitine and acylcarnitine profile. J. Inherit. Metab. Dis. 24, 51–59. Spiekerkoetter, U., Tokunaga, C., Wendel, U., Mayatepek, E., Exil, V., Duran, M., Wijburg, F.A., Wanders, R.J.A., Strauss, A.W., 2004. Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress. Eur. J. Clin. Invest. 34, 191– 196. Stanley, W.C., Lopaschuk, G.D., Hall, J.L., McCormack, J.G., 1997. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions––potential for pharmacological interventions. Cardiovasc. Res. 33, 243–257. Steiber, A.L., Weatherspoon, L.J., Spry, L., Davis, A.T., 2004. Serum carnitine concentrations correlated to clinical outcome parameters in chronic hemodialysis patients. Clin. Nutr. 23, 27–34. Tahiliani, A.G., Keene, T., Kaplan, R.S., 1992. Characterization of the inhibitor sensitivity of the coenzyme-a transport-system in isolated rat-heart mitochondria. J. Bioenerg. Biomembr. 24, 635–640. Tanaka, M., Nakamura, F., Mizokawa, S., Matsumura, A., Matsumura, K., Watanabe, Y., 2003. Role of acetyl-L -carnitine in the brain: revealed by bioradiography. Biochem. Biophys. Res. Commun. 306, 1064–1069. Terada, R., Matsubara, T., Koh, N., Nakamura, J., Hotta, N., 1998. Effects of propionyl-L -carnitine on cardiac dysfunction in streptozotocin-diabetic rats. Eur. J. Pharmacol. 357, 185–191. Tomassini, V., Pozzilli, C., Onesti, E., Pasqualetti, P., Marinelli, F., Pisani, A., Fieschi, C., 2004. Comparison of the effects of acetyl L -carnitine and amantadine for the treatment of fatigue in multiple sclerosis: results of a pilot, randomised, double-blind, crossover trial. J. Neurol. Sci. 218, 103–108. Tran, T.N., Christophersen, B.O., 2001. Studies on the transport of acetyl groups from peroxisomes to mitochondria in isolated liver cells oxidizing the polyunsaturated fatty acid 22: 4n-6. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1533, 255–265. van der Leij, F.R., Huijkman, N.C.A., Boomsma, C., Kuipers, J.R.G., Bartelds, B., 2000. Genomics of the human carnitine acyltransferase genes. Mol. Genet. Metab. 71, 139–153. Vanella, A., Russo, A., Acquaviva, R., Campisi, A., Di Giacomo, C., Sorrenti, V., Barcellona, M.L., 2000. L -Propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector. Cell Biol. Toxicol. 16, 99–104. Vermeulen, R.C.W., Scholte, H.R., 2004. Exploratory open label, randomized study of acetyl- and propionylcarnitine in chronic fatigue syndrome. Psychosom. Med. 66, 276–282. Vessey, D.A., Chen, W., Ramsay, R.R., 1991. Effect of carboxylic-acid xenobiotics and their metabolites on the activity of carnitine acyltransferases. Biochem. J. 279, 895–897. Winter, S.C., Buist, N.R.M., 2000. Cardiomyopathy in childhood, mitochondrial dysfunction, and the role of L -carnitine. Am. Heart J. 139, S63–S69. Wiseman, L.R., Brogden, R.N., 1998. Propionyl-L -carnitine. Drugs Aging 12, 243–248. Yamaguti, K., Kuratsune, H., Watanabe, Y., Takahashi, M., Nakamoto, I., Machii, T., Jacobsson, G., Onoe, H., Matsumura, K., Valind, S., Langstrom, B., Kitani, T., 1996. Acylcarnitine metabolism during fasting and after refeeding. Biochem. Biophys. Res. Commun. 225, 740–746. Yaris, N., Akyuz, C., Coskun, T., Buyukpamukcu, M., 2002. Serum carnitine levels of pediatric cancer patients. Pediatr. Hematol. Oncol. 19, 1–8. Zammit, V.A., 1998. Role of malonyl-CoA-sensitive carnitine palmitoyltransferase in the aetiology and treatment of some metabolic complications of diabetes. Diabetes Nutr. Metab. 11, 200–211.