Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Geraniol attenuates 4NQO-induced tongue carcinogenesis through downregulating the activation of NF-κB in rats

Diabetic cardiomyopathy is preceded by mitochondrial alterations, and progresses to heart failure. We studied whether treatment with methylene blue (MB), a compound that was reported to serve as an alternate electron carrier within the mitochondrial electron transport chain (ETC), improves mitochondrial metabolism and cardiac function in type 1 diabetes. MB was administered at 10 mg/kg/day to control and diabetic rats. Both echocardiography and hemodynamic studies were performed to assess cardiac function. Mitochondrial studies comprised the measurement of oxidative phosphorylation and specific activities of fatty acid oxidation enzymes. Proteomic studies were employed to compare the level of lysine acetylation on cardiac mitochondrial proteins between the experimental groups. We found that MB facilitates NADH oxidation, increases NAD⁺, and the activity of deacetylase Sirtuin 3, and reduces protein lysine acetylation in diabetic cardiac mitochondria. We identified that lysine acetylation on 83 sites in 34 proteins is lower in the MB-treated diabetic group compared to the same sites in the untreated diabetic group. These changes occur across critical mitochondrial metabolic pathways including fatty acid transport and oxidation, amino acid metabolism, tricarboxylic acid cycle, ETC, transport, and regulatory proteins. While the MB treatment has no effect on the activities of acyl-CoA dehydrogenases, it decreases 3-hydroxyacyl-CoA dehydrogenase activity and long-chain fatty acid oxidation, and improves cardiac function. Providing an alternative route for mitochondrial electron transport is a novel therapeutic approach to decrease lysine acetylation, alleviate cardiac metabolic inflexibility, and improve cardiac function in diabetes.     

Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its co-enzyme A and carnitine esters

1. The CoA and carnitine esters of 2-bromopalmitate are extremely powerful and specific inhibitors of mitochondrial fatty acid oxidation. 2. 2-Bromopalmitoyl-CoA, added as such or formed from 2-bromopalmitate, inhibits the carnitine-dependent oxidation of palmitate or palmitoyl-CoA, but not the oxidation of palmitoylcarnitine, by intact liver mitochondria. 3. 2-Bromopalmitoylcarnitine inhibits the oxidation of palmitoylcarnitine as well as that of palmitate or palmitoyl-CoA. It has no effect on succinate oxidation, but inhibits that of pyruvate, 2-oxoglutarate or hexanoate; however, the oxidation of these substrates (but not of palmitate, palmitoyl-CoA or palmitoyl-carnitine) is restored by carnitine. 4. In damaged mitochondria, added 2-bromopalmitoyl-CoA does inhibit palmitoylcarnitine oxidation; pyruvate oxidation is unaffected by the inhibitor alone, but is impaired if palmitoylcarnitine is subsequently added. 5. The findings have been interpreted as follows. 2-Bromopalmitoyl-CoA inactivates (in a carnitine-dependent manner) a pool of carnitine palmitoyltransferase which is accessible to external acyl-CoA. This results in inhibition of palmitate or palmitoyl-CoA oxidation. A second pool of carnitine palmitoyltransferase, inaccessible to added acyl-CoA in intact mitochondria, can generate bromopalmitoyl-CoA within the matrix from external 2-bromopalmitoylcarnitine; this reaction is reversible. Such internal 2-bromopalmitoyl-CoA inactivates long-chain beta-oxidation (as does added 2-bromopalmitoyl-CoA if the mitochondria are damaged) and its formation also sequesters intramitochondrial CoA. Since this CoA is shared by pyruvate and 2-oxoglutarate dehydrogenases, the oxidation of their substrates is depressed by 2-bromopalmitoylcarnitine, unless free carnitine is available to act as a ;sink' for long-chain acyl groups. 6. These effects are compared with those reported for other inhibitors of fatty acid oxidation.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Cardiac Metabolic Pathways Affected in the Mouse Model of Barth Syndrome

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F₀F₁-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.     

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

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.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.     

Lipid oxidation by heart mitochondria from young adult and senescent rats

1. State-3 (i.e. ADP-stimulated) rates of O(2) uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O(2) uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[(3)H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O(2) uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O(2) uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O(2) uptake with palmitoylcarnitine in senescence is discussed.     

Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.     

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP.

The present study on saponin-treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl-CoA+carnitine, palmitoyl-L-carnitine and octanoyl-L-carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K(m) of oxidative phosphorylation for ADP. Octanoyl-D-carnitine, as well as palmitate, palmitoyl-CoA, and palmitoyl-L-carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.     

Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds

We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments.When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine, Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation.In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate.We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level.These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation. (Mol Cell Biochem 174: 61–66, 1997)     

Study of some factors controlling fatty acid oxidation in liver mitochondria of obese Zucker rats.

Livers of genetically obese Zucker rats showed, compared with lean controls, hypertrophy and enrichment in triacylglycerols, indicating that fatty acid metabolism was directed towards lipogenesis and esterification rather than towards fatty acid oxidation. Mitochondrial activities of cytochrome c oxidase and monoamine oxidase were significantly lower when expressed per g wet wt. of liver, whereas peroxisomal activities of urate oxidase and palmitoyl-CoA-dependent NAD+ reduction were unchanged. Liver mitochondria were able to oxidize oleic acid at the same rate in both obese and lean rats. For reactions occurring inside the mitochondria, e.g. octanoate oxidation and palmitoyl-CoA dehydrogenase, no difference was found between both phenotypes. Total carnitine palmitoyl-, octanoyl- and acetyl-transferase activities were slightly higher in mitochondria from obese rats, whereas the carnitine content of both liver tissue and mitochondria was significantly lower in obese rats compared with their lean littermates. The carnitine palmitoyltransferase I activity was slightly higher in liver mitochondria from obese rats, but this enzyme was more sensitive to malonyl-CoA inhibition in obese than in lean rats. The above results strongly suggest that the impaired fatty acid oxidation observed in the whole liver of obese rats is due to the diminished transport of fatty acids across the mitochondrial inner membrane via the carnitine palmitoyltransferase I. This effect could be reinforced by the decreased mitochondrial content per g wet wt. of liver. The depressed fatty acid oxidation may explain in part the lipid infiltration of liver observed in obese Zucker rats.     

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.     

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

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Metabolic effects of L-carnitine on prepubertal rat Sertoli cells.

The role of carnitine on Sertoli cell metabolism was investigated. Carnitine effects on Sertoli cell lipid metabolism were evaluated by measuring the intracellular levels of non-esterified fatty acids (NEFA) and ketone bodies. The concentration of NEFA in Sertoli cell cultured in the presence of carnitine is significantly reduced as compared to control, while, no significant changes were observed in the concentration of ketone bodies. The functional parameters evaluated to assess the influence of carnitine on Sertoli cell carbohydrate metabolism, i.e., lactate and pyruvate production, lactate dehydrogenase activity and hexose transport, were all significantly increased following carnitine in vitro supplementation. Thus, carnitine appears to drive Sertoli cell intermediary metabolism in an intimately interrelated way, stimulating both fatty acid breakdown and glycolysis. Our results indicate that Sertoli cells are a possible target for a widespread metabolic action of carnitine and strongly support the involvement of carnitine in the regulation of Sertoli cell functions which are related with germ cell "nutrition", convincingly suggesting a direct influence of the compound at testis level.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.