Chymotrypsin activates cardiac mitochondrial carnitine-acylcarnitine translocase.

The carnitine-acylcarnitine translocase facilitates carnitine and acylcarnitine transport into the mitochondrial matrix during beta-oxidation. Our results demonstrate that chymotrypsin can activate the maximal velocity of N-ethylmaleimide (NEM)-sensitive carnitine or palmitoylcarnitine exchange 7-fold, while doubling the affinity of the translocase for carnitine. Chymotrypsin activation is strictly dependent on the presence of free or short-chain acylcarnitine in the proteolysis medium, the extent of activation decreasing as the acylcarnitine chain length in the proteolysis medium increases. Chymotrypsin treatment decreases the apparent I50 value (inhibitor concentration required to give half-maximal inhibition) of the translocase for inhibition by NEM only under conditions which produce translocase activation. Modification of submitochondrial particle membranes by chymotrypsin does not result in gross ultrastructural changes or in an increase in the passive permeability of these membranes to carnitine. The data suggest that carnitine binding produces a change in translocase conformation which allows chymotrypsin modification to occur. This modification alters the kinetic and inhibitor-binding properties of the translocase.     

Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism.

Monitoring of the exchange-diffusion of carnitine, acetylcarnitine and ADP by measuring the influx of radioactive substrates into mitochondria or their efflux, as commonly employed, underestimated their true transport. Higher transport rates were realized when the imports were monitored by analysing, in the entire incubation medium, formation of metabolites that could proceed only after the substrate import. A recycling of substrate present in an inner microenvironment near the translocase and in the external medium appeared to be responsible for these results. Microcompartmentation of carnitine was observable also at 30 degrees C. These findings strengthen the concept that a sharing of a microcompartment between transporters and enzymes metabolizing the entered substrates occurs and appears to offer a kinetic advantage for the reactions involved. The possibility that different segments of metabolism involving the same substrate may proceed at different loci within the matrix and thus be amenable to independent controls is also indicated by these findings.     

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.     

Effect of carnitine on mitochondrial oxidation of palmitoylcarnitine

The effects of carnitine on the metabolism of palmitoylcarnitine were studied by using isolated rat liver mitochondria. Particular attention was given to carnitine acyltransferase-mediated interactions between carnitine and the mitochondrial CoA pool. Carnitine concentrations less than 1.25mm resulted in an increased production of acetylcarnitine during palmitoylcarnitine oxidation. Despite this shunting of C₂ units to acetylcarnitine formation, no change was observed in the rate of oxygen consumption or major product formation (citrate or acetoacetate). Further, no changes were observed in the mitochondrial content of acetyl-CoA, total acid-soluble CoA or acid-insoluble acyl-CoA. These observations support the concept, based on studies in vivo, that the carnitine/acylcarnitine pool is metabolically sluggish and the acyl-group flux low as compared with the CoA/acyl-CoA pool. Acid-insoluble acyl-CoA content was decreased and CoA content increased at carnitine concentrations greater than 1.25mm. When [¹⁴C]carnitine was used in the incubations, it was demonstrated that this resulted from acid-insoluble acylcarnitine formation from intramitochondrial acid-insoluble acyl-CoA mediated by carnitine palmitoyltransferase B. Again, the higher carnitine concentrations resulted in no changes in the rates of oxygen consumption or major product formation. The above effects of carnitine were observed whether citrate or acetoacetate was the major product of oxidation. In contrast, an increase in acetyl-CoA concentration was observed at high carnitine concentrations only when acetoacetate was the product. Since the rate of acetoacetate production was not changed, these higher acetyl-CoA concentrations suggest that a new steady state had been established to maintain acetoacetate-production rates. Since there was no change in acetyl-CoA concentration when citrate was the major product, a change in the activity of the pathway utilizing acetyl-CoA for ketone-body synthesis and the potential regulation of this pathway must be considered.     

Solubilization and reconstitution of rat liver mitochondrial carnitine acylcarnitine translocase

Carnitine acylcarnitine translocase has been solubilized from inverted inner membrane vesicles of rat liver mitochondria with octyl glucoside and reconstituted into asolectin liposomes. For both processes, optimization of the detergent to phospholipid ratio was found crucial for obtaining reconstitutively active liposomes. Reassembly of the solubilized carrier into asolectin liposomes was achieved either by the octyl glucoside dilution method or by Extracti-Gel D column chromatography. The reconstituted system catalyzed exchange diffusion of carnitine, exhibited the expected inhibitor and temperature sensitivity, and discriminated between stereoisomers of octanoylcarnitine. The activity of unidirectional import of carnitine was low compared to exchange diffusion. It showed high-temperature sensitivity and a loss of activity on prolonged sonication that was regained by an appropriate freeze-thaw step subsequently.     

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.     

Relationship between acid-soluble carnitine and coenzyme A pools in vivo

The relationship between the acid-soluble carnitine and coenzyme A pools was studied in fed and 24-h-starved rats after carnitine administration. Carnitine given by intravenous injection at a dose of 60μmol/100g body wt. was integrated into the animal's endogenous carnitine pool. Large amounts of acylcarnitines appeared in the plasma and liver within 5min of carnitine injection. Differences in acid-soluble acylcarnitine concentrations were observed between fed and starved rats after injection and reflected the acylcarnitine/carnitine relationship seen in the endogenous carnitine pool of the two metabolic states. Thus, a larger acylcarnitine production was seen in starved animals and indicated a greater source of accessible acyl-CoA molecules. In addition to changes in the amount of acylcarnitines present, the specific acyl groups present also varied between groups of animals. Acetylcarnitine made up 37 and 53% of liver acid-soluble acylcarnitines in uninjected fed and starved animals respectively. At 5min after carnitine injection hepatic acid-soluble acylcarnitines were 41 and 73% in the form of acetylcarnitine in fed and starved rats respectively. Despite these large changes in carnitine and acylcarnitines, no changes were observed in plasma non-esterified fatty acid or β-hydroxybutyrate concentrations in either fed or starved rats. Additionally, measurement of acetyl-CoA, coenzyme A, total acid-soluble CoA and acid-insoluble CoA demonstrated that the hepatic CoA pool was resistant to carnitine-induced changes. This lack of change in the hepatic CoA pool or ketone-body production while acyl groups are shunted from acyl-CoA molecules to acylcarnitines suggests a low flux through the carnitine pool compared with the CoA pool. These results support the concept that the carnitine/acid-soluble acylcarnitine pool reflects changes in, rather than inducing changes in, the hepatic CoA/acyl-CoA pool.     

Aggregation of submitochondrial particles by heparin and its application to the study of carnitine transport.

A novel technique for the separation of submitochondrial particles from the external medium, an essential procedure in transport studies, was devised. Very low concentrations of heparin (5-10 micrograms/ml) aggregate the particles and permit their rapid sedimentation in a micro-centrifuge. The transfer of activated fatty acids into mitochondria for oxidation depends on the exchange of matrix carnitine for external fatty-acylcarnitine. To study the matrix face of the carnitine/acylcarnitine translocase, inverted submitochondrial particles were prepared and loaded with L-[14C]carnitine. As found in intact mitochondria, the Km value for L-carnitine was 8 mM, that for palmitoyl-L-carnitine was two orders of magnitude lower, and 11-trimethylaminoundecanoyl-DL-carnitine was a competitive inhibitor. The properties of the carrier exposed to the outer and to the matrix sides of the mitochondrial inner membrane are thus similar.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

tRNA import across the mitochondrial inner membrane in T. brucei requires TIM subunits but is independent of protein import

Mitochondrial tRNA import is widespread, but mechanistic insights of how tRNAs are translocated across mitochondrial membranes remain scarce. The parasitic protozoan T. brucei lacks mitochondrial tRNA genes. Consequently, it imports all organellar tRNAs from the cytosol. Here we investigated the connection between tRNA and protein translocation across the mitochondrial inner membrane. Trypanosomes have a single inner membrane protein translocase that consists of three heterooligomeric submodules, which all are required for import of matrix proteins. In vivo depletion of individual submodules shows that surprisingly only the integral membrane core module, including the protein import pore, but not the presequence-associated import motor are required for mitochondrial tRNA import. Thus we could uncouple import of matrix proteins from import of tRNAs even though both substrates are imported into the same mitochondrial subcompartment. This is reminiscent to the outer membrane where the main protein translocase but not on-going protein translocation is required for tRNA import. We also show that import of tRNAs across the outer and inner membranes are coupled to each other. Taken together, these data support the ‘alternate import model’, which states that tRNA and protein import while mechanistically independent use the same translocation pores but not at the same time.     

Hepatic release of carnitine: effect of increased concentration by clofibrate treatment.

The release of carnitine is an important metabolic function of the liver. In the present study, we have investigated the effect of increased carnitine concentration on the hepatic release of carnitine. Hepatic carnitine concentration was increased in rats by clofibrate treatment. Release of carnitine was investigated as its efflux from perfused liver and its secretion into bile. A significantly smaller proportion of the hepatic pool of carnitine was released into the perfusion medium when carnitine concentration was increased by clofibrate treatment. However, the amount of carnitine released (nmol/g liver) was comparable to that of control rats. Increased carnitine concentration by clofibrate treatment also did not affect the rate of biliary secretion of carnitine. In control rats, nearly 50% of the released carnitine, in both the perfusion medium and bile, was acylcarnitine whereas in clofibrate-treated rats 35% of the released carnitine was acylcarnitine. Release into the perfusion medium was the major route for the hepatic export of carnitine. We conclude that when hepatic carnitine concentration is increased by clofibrate treatment, a smaller proportion of the hepatic carnitine pool is released, but the amount of carnitine released (nmol/g liver) is not greatly different than that from control animals.     

Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as ³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.     

Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.

In Saccharomyces cerevisiae, beta-oxidation of fatty acids is confined to peroxisomes. The acetyl-CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO(2) and H(2)O. Two pathways for the transport of acetyl-CoA to the mitochondria have been proposed. The first involves peroxisomal conversion of acetyl-CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria. The second pathway involves peroxisomal conversion of acetyl-CoA into acetylcarnitine, which is subsequently transported to the mitochondria. Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine-dependent acetyl-CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups. The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library. In addition to the previously reported carnitine acetyl-CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane.     

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.     

Primary defect of juvenile visceral steatosis (jvs) mouse with systemic carnitine deficiency is probably in renal carnitine transport system

We investigated the reabsorptional system for carnitine in the kidney to elucidate the mechanism of carnitine deficiency in juvenile visceral steatosis (jvs) mice. Jvs mice had a higher rate of carnitine excretion at 10 days after birth than the controls, in spite of having no pathological acylcarnitine in the urine. In an experiment to assay the uptake of carnitine using kidney slices, homozygous mutants showed significantly lower rates of Na-dependent carnitine uptake than controls. Heterozygous mice showed values of transport activity intermediate between homozygous mutants and homozygous controls. Scatchard plots (transport activity versus transport activity/carnitine concentration) revealed that the homozygous mutants had a defect in the hihg affinity site (K_m = 58 μM) in the Na-dependent carnitine transport system in the kidney. These results indicate that the primary defect of jvs mice is most probably related to the system for reabsorption of carnitine in the kidney.     

An essential requirement of cardiolipin for mitochondrial carnitine acylcarnitine translocase activity. Lipid requirement of carnitine acylcarnitine translocase

The phospholipid requirement for the optimal solubilization of carnitine acylcarnitine translocase from the inner membrane vesicles of rat liver mitochondria and for its reconstitution in liposomes was investigated. At the octylglucoside-solubilization step, the presence of cardiolipin proved superior to the other lipids tested. For reconstitution, a mixture having phosphatidylcholine, phosphatidylethanolamine and cardiolipin was found to be particularly effective. The requirement of cardiolipin at this step was met less effectively by other anionic phospholipids. Moreover, in intact mitochondria of rat liver and heart, the translocase activity was markedly inhibited by micromolar concentrations of doxorubicin, a specific cardiolipin-binding agent.     

Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier

The submitochondrial and subcellular distributions of the carnitine-acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high-abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti-peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.