Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD⁺. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V _max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

The mitochondrial carnitine/acylcarnitine carrier: function, structure and physiopathology

The carnitine/acylcarnitine carrier (CAC) is a transport protein of the inner mitochondrial membrane that belongs to the mitochondrial carrier protein family. In its cytosolic conformation the carrier consists of a bundle of six transmembrane α-helices, which delimit a water filled cavity opened towards the cytosol and closed towards the matrix by a network of interacting charged residues. Most of the functional data on this transporter come from studies performed with the protein purified from rat liver mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The carnitine/acylcarnitine carrier transports carnitine and acylcarnitines with acyl chains of various lengths from 2 to 18 carbon atoms. The mammalian transporter exhibits higher affinity for acylcarnitines with longer carbon chains. The functional data indicate that CAC plays the important function of catalyzing transport of acylcarnitines into the mitochondria in exchange for intramitochondrial free carnitine. This results in net transport of fatty acyl units into the mitochondrial matrix where they are oxidized by the β-oxidation enzymes. The essential role of the transporter in cell metabolism is demonstrated by the fact that alterations of the human gene SLC25A20 coding for CAC are associated with a severe disease known as carnitine carrier deficiency. This autosomal recessive disorder is characterized by life-threatening episodes of coma induced by fasting, cardiomyopathy, liver dysfunction, muscle weakness, respiratory distress and seizures. Until now 35 different mutations of CAC gene have been identified in carnitine carrier deficient patients. Some missense mutations concern residues of the signature motif present in all mitochondrial carriers. Diagnosis of carnitine carrier deficiency requires biochemical and genetic tests; treatment is essentially limited to important dietetic measures. Recently, a pharmacological approach based on the use of statins and/or fibrates for the treatment of CAC-deficient patients with mild phenotype has been proposed.     

Molecular Mechanism of Inhibition of the Mitochondrial Carnitine/Acylcarnitine Transporter by Omeprazole Revealed by Proteoliposome Assay,Mutagenesis and Bioinformatics

The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC₅₀ of the inhibition of the WT and the C-less CACT by omeprazole were 5.4 µM and 29 µM, respectively. Inhibition kinetics showed non competitive inhibition of the WT and competitive inhibition of the C-less. The presence of carnitine or acylcarnitines during the incubation of the proteoliposomes with omeprazole increased the inhibition. Using site-directed Cys mutants it was demonstrated that C283 and C136 were essential for covalent inhibition. Molecular docking of omeprazole with CACT indicated the formation of both covalent interactions with C136 and C283 and non-covalent interactions in agreement with the experimental data.     

A novel mitochondrial carnitine-acylcarnitine translocase induced by partial hepatectomy and fasting

The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism. In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT). Here we report a novel mammalian protein homologous to CACT. The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity. The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed. In addition, CACL is induced by partial hepatectomy or fasting. Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.     

Interaction of mildronate with the mitochondrial carnitine/acylcarnitine transport protein

The interaction of mildronate [3-(2,2,2-trimethylhydrazine) propionate] with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Mildronate, externally added to the proteoliposomes, strongly inhibited the carnitine/carnitine antiport catalyzed by the reconstituted transporter with an IC(50) of 560 muM. A kinetic analysis revealed that the inhibition is completely competitive, that is, mildronate interacts with the substrate-binding site. The half-saturation constant of the transporter for external mildronate (K(i)) is 530 muM. Carnitine/mildronate antiport has been measured as [(3)H]carnitine uptake into proteoliposomes containing internal mildronate or as [(3)H]carnitine efflux from proteoliposomes in the presence of external mildronate, indicating that mildronate is transported by the carnitine/acylcarnitine transporter and that the inhibition observed was due to the transport of mildronate in the place of carnitine. The intraliposomal half-saturation constant for mildronate transport (K(m)) has been determined. Its value, 18 mM, is much higher than the external half-saturation constant (K(i)) in agreement with the asymmetric properties of the transporter. In vivo, the antiport reaction between cytosolic (administered) mildronate and matrix carnitine may cause intramitochondrial carnitine depletion. This effect, together with the inhibition of the physiological transport, will lead to impairment of fatty acid utilization.     

Carnitine-acylcarnitine translocase deficiency, clinical, biochemical and genetic aspects

The carnitine–acylcarnitine translocase (CACT) is one of the components of the carnitine cycle. The carnitine cycle is necessary to shuttle long-chain fatty acids from the cytosol into the intramitochondrial space where mitochondrial β-oxidation of fatty acids takes place. The oxidation of fatty acids yields acetyl-coenzyme A (CoA) units, which may either be degraded to CO₂ and H₂O in the citric acid cycle to produce ATP or converted into ketone bodies which occurs in liver and kidneys.

Metabolic consequences of a defective CACT are hypoketotic hypoglycaemia under fasting conditions, hyperammonemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine and an abnormal acylcarnitine profile with marked elevation of the long-chain acylcarnitines.

Clinical signs and symptoms in CACT deficient patients, are a combination of energy depletion and endogenous toxicity. The predominantly affected organs are brain, heart and skeletal muscle, and liver, leading to neurological abnormalities, cardiomyopathy and arrythmias, skeletal muscle damage and liver dysfunction. Most patients become symptomatic in the neonatal period with a rapidly progressive deterioration and a high mortality rate. However, presentations at a later age with a milder phenotype have also been reported.

The therapeutic approach is the same as in other long-chain fatty acid disorders and includes intravenous glucose (± insulin) administration to maximally inhibit lipolysis and subsequent fatty acid oxidation during the acute deterioration, along with other measures such as ammonia detoxification, depending on the clinical features. Long-term strategy consists of avoidance of fasting with frequent meals and a special diet with restriction of long-chain fatty acids. Due to the extremely low free carnitine concentrations, carnitine supplementation is often needed.

Acylcarnitine profiling in plasma is the assay of choice for the diagnosis at a metabolite level. However, since the acylcarnitine profile observed in CACT-deficient patients is identical to that in CPT2-deficient patients, definitive identification of CACT-deficiency in a certain patient requires determination of the activity of CACT. Subsequently, mutational analysis of the CACT gene can be performed. So far, 9 different mutations have been identified in the CACT gene.     

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.     

Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.     

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist

In the brain β-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial β-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B^0,+ and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor α (PPARα), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter—OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARα activator–2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARα stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.     

Functional analysis of mutant human carnitine acylcarnitine translocases in yeast

Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient approximately 27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.     

Differential carnitine/acylcarnitine translocase expression defines distinct metabolic signatures in skeletal muscle cells

Import of acylcarnitine into mitochondrial matrix through carnitine/acylcarnitine-translocase (CACT) is fundamental for lipid catabolism. To probe the effect of CACT down-expression on lipid metabolism in muscle, human myocytes were stably transfected with CACT-antisense construct. In presence of low concentration of palmitate, transfected cells showed decreased palmitate oxidation and acetyl-carnitine content, increased palmitoyl-carnitine level, and reduced insulin-dependent decrease of fatty acylcarnitine-to-fatty acyl-CoA ratio. The augmented palmitoyl-carnitine synthesis, also in the presence of insulin, could be related to an altered regulation of carnitine-palmitoyl-transferase 1 (CPT 1) by malonyl-CoA, whose synthesis is dependent by the availability of cytosolic acetyl-groups. Indeed, all the described effects were completely overcome by CACT neo-expression by recombinant adenovirus vector or by addition of acetyl-carnitine to cultures. Acetyl-carnitine effect was related to an increase of malonyl-CoA and was abolished by down-expression, via antisense RNA strategy, of acetyl-CoA carboxylase-beta, the mitochondrial membrane enzyme involved in the direct CPT 1 inhibition via malonyl-CoA synthesis. Thus, in our experimental model the modulation of CACT expression has consequences for CPT 1 activity, while the biologic effects of acetyl-carnitine are not associated with a generic supply of energy compounds but to the anaplerotic property of the molecule.     

Carnitine Insufficiency Caused by Aging and Overnutrition Compromises Mitochondrial Performance and Metabolic Control

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine dimunition was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete β-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.Disturbances in mitochondrial genesis, morphology, and function are increasingly recognized as components of insulin resistance and the metabolic syndrome (1–3). Still unclear is whether poor mitochondrial performance is a predisposing factor or a consequence of the disease process. The latter view is supported by recent animal studies linking diet-induced insulin resistance to a dysregulated mitochondrial phenotype in skeletal muscle, marked by excessive β-oxidation, impaired substrate switching during the fasted to fed transition, and coincident reduction of organic acid intermediates of the tricarboxylic acid cycle (4, 5). In these studies, both diet-induced and genetic forms of insulin resistance were specifically linked to high rates of incomplete fat oxidation and intramuscular accumulation of fatty acylcarnitines, byproducts of lipid catabolism that are produced under conditions of metabolic stress (5, 6). Most compelling, we showed that genetically engineered inhibition of fat oxidation lowered intramuscular acylcarnitine levels and preserved glucose tolerance in mice fed a high fat diet (5, 7). In aggregate, the findings established a strong connection between mitochondrial bioenergetics and insulin action while raising new questions regarding the roles of incomplete β-oxidation and acylcarnitines as potential biomarkers and/or mediators of metabolic disease.In another recent investigation we found that oral carnitine supplementation improved insulin sensitivity in diabetic mice, in parallel with a marked rise in plasma acylcarnitines (8). This occurred in three distinct models of glucose intolerance; aging, genetic diabetes, and high fat feeding (8). The antidiabetic actions of carnitine were accompanied by an increase in whole body glucose oxidation, a surprising result given that carnitine is best known for its essential role in permitting mitochondrial translocation and oxidation of long chain acyl-CoAs. Carnitine palmitoyltransferase 1 (CPT1)² executes the initial step in this process by catalyzing the reversible transesterification of long chain acyl-CoA with carnitine. The long chain acylcarnitine (LCAC) product of CPT1 traverses the inner membrane via carnitine/acylcarnitine translocase (CACT) and is then delivered to CPT2, which regenerates acyl-CoA on the matrix side of the membrane where β-oxidation occurs. Notably, however, in addition to its requisite role in fatty acid oxidation, carnitine also facilitates mitochondrial efflux of excess carbon fuels. Thus, in the event that rates of substrate catabolism exceed energy demand, accumulating acyl-CoA intermediates are converted back to acylcarnitines, which can then exit the organelle and the tissue. This aspect of carnitine function has remained relatively understudied.The finding that carnitine supplementation improved glucose tolerance while increasing circulating acylcarnitines favors the interpretation that production and efflux of these metabolites is beneficial rather than detrimental (9, 10). Thus, at present, we view these metabolites as biomarkers rather than mediators of metabolic dysfunction. Acylcarnitine accumulation in insulin-resistant skeletal muscles might reflect a failed attempt to combat “mitochondrial stress” and/or an impediment in tissue export; either of which could arise should availability of free carnitine become limiting. Fitting with this scenario, we postulated that carnitine insufficiency might contribute to mitochondrial dysfunction and insulin resistance. To address this possibility carnitine homeostasis was examined in rodent models of obesity, diabetes, and aging. Our results show that chronic metabolic stress does indeed compromise whole body carnitine status. Low carnitine levels in severely obese rats were associated with aberrant mitochondrial fuel metabolism, whereas oral carnitine supplementation reversed these perturbations in concert with improved glucose tolerance and increased acylcarnitine efflux. Complementary studies in primary human myocytes suggest that the therapeutic actions of carnitine are mediated in part through carnitine acetyltransferase (CrAT), a mitochondrial matrix enzyme that promotes glucose disposal. These findings underscore the multifaceted roles of the carnitine shuttle system, not only in permitting β-oxidation but also for maintaining mitochondrial performance and glucose homeostasis in the face of energy surplus.     

Tryptophan 224 of the rat mitochondrial carnitine/acylcarnitine carrier is crucial for the antiport mechanism

The mitochondrial carnitine/acylcarnitine carrier (CACT) catalyzes an antiport of carnitine and acylcarnitines and also a uniport reaction with a rate of about one tenth with respect to the antiport rate. The antiport process results from the coupling of the two uniport reactions in opposite directions. In this mechanism, the transition of the carrier from the outward open conformation to the inward open one (or vice versa) is much faster for the carrier-substrate complex than for the unbound carrier. To investigate the molecular determinants that couple the binding of the substrate with the conformational transitions, site directed mutagenesis has been employed. The antiport or the uniport reaction was followed as [³H]carnitine uptake in or efflux from proteoliposomes reconstituted with the WT or Trp mutants of the rat CACT. Substitution of each the three Trp residues led to different results. Nearly no variations were observed upon substitution of W192 and/or W296 with Ala. While, substantial alteration of the transport function was observed in the mutants W224A, W224Y and W224F. Mutation of W224 led to the loss of the antiport function while the uniport function was unaltered. In these mutants impairment of the substrate affinity on the external side was also observed. The data highlights that W224 is involved in the coupling of the substrate binding with the matrix gate opening. The experimental data are in line with predictions by homology modeling of the CACT in its cytosolic (c-state) or matrix (m-state) opened conformations.     

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Ketogenesis in isolated rat liver mitochondria. II. Factors affecting the rate of β-oxidation

1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium (l-carnitine, CoASH, hexokinase, albumin).2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine.3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine.4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C).5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate.6. l-Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids.7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions.8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C).9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.     

Interaction of beta-lactam antibiotics with the mitochondrial carnitine/acylcarnitine transporter

The interaction of beta-lactams with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Cefonicid, cefazolin, cephalothin, ampicillin, piperacillin externally added to the proteoliposomes, inhibited the carnitine/carnitine antiport catalysed by the reconstituted transporter. The most effective inhibitors were cefonicid and ampicillin with IC50 of 6.8 and 7.6mM, respectively. The other inhibitors exhibited IC50 values above 36 mM. Kinetic analysis performed with cefonicid and ampicillin revealed that the inhibition is completely competitive, i.e., the inhibitors interact with the substrate binding site. The Ki of the transporter is 4.9 mM for cefonicid and 9.9 mM for ampicillin. Cefonicid inhibited the transporter also on its internal side. The IC50 was 12.9 mM indicating that the inhibition was less pronounced than on the external side. Ampicillin and the other inhibitors were much less effective on the internal side. The beta-lactams were not transported by the carnitine/acylcarnitine transporter. Cephalosporins, and at much lower extent penicillins, caused irreversible inhibition of the transporter after prolonged time of incubation. The most effective among the tested antibiotics was cefonicid with IC50 of 0.12 mM after 60 h of incubation. The possible in vivo implications of the interaction of the beta-lactam antibiotics with the transporter are discussed.     

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C_16:0+C_18:1)/C₂. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.     

Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation

Background

The mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches.

Methods

The effect of GSH and GSSG on the [³H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody.

Results

GSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37 °C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC.

Conclusions

CAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation.

General significance

CAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.