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International audience; Carnitine palmitoyltransferase 2 (CPTII) deficiency is among the most common inborn errors of mitochondrial fatty acid ␤-oxidation (FAO). Clinical phenotype varies in relation to the metabolic block, as assessed by studies of FAO in patient fibroblasts. Thus, fibroblasts from patients with mild manifestations have appreciable residual CPTII enzyme activity, in contrast to those from severely affected patients. In the present study, we hypothesized that the hypolipidemic drug bezafibrate, acting as an activator of the peroxisome proliferator-activated receptor ␣ might stimulate FAO in CPTII-deficient cells. Data obtained show that bezafibrate treatment of mild-type CPTII-deficient cells resulted in a time-and dose-dependant increase in CPTII mRNA (from ϩ47% to ϩ66%) and residual enzyme activity (from ϩ54% to 135%), and led to normalization of 3 H-palmitate and 3 H-myristate cellular oxidation rates. Bezafibrate did not correct FAO in fibroblasts from patients with severe phenotype. This study establishes for the first time that peroxisome prolif-erator-activated receptor activators, acting via stimulation of gene expression, can stimulate CPTII residual activity to a level sufficient to allow normal FAO flux in deficient human fibro-blasts, and suggests that this approach should be tested in other inborn errors of mitochondrial ␤-oxidation. (Pediatr Res 54: 446-451, 2003)

International audience; The mitochondrial oxidation of fatty acids (FAO) is the main energy-producing pathway in skeletal and cardiac muscle. Starting from standard muscle biopsies (100-200 mg), we determined the optimal conditions of mitochondrial oxygen consumption by the FAO pathway, and in parallel we performed the isolation and primary culture of muscle cells to test their cellular FAO capacities. The determinations of maximal b-oxidation rates in the presence of palmitoyl-CoA or palmitoyl-L L-carnitine (mean AE SEM: 32:5 AE 2:0 and 34:1 AE 1:3 nmol O 2 min À1 mg À1 protein, n ¼ 16, respectively) provide a screening method of mitochondrial fatty acid transport system and intra-mitochondrial b-oxidation. We also determined the conditions of tritiated palmitate oxidation by human myoblasts (mean AE SEM: 6:6 AE 0:1 nmol 3 H fatty acid h À1 mg À1 protein, n ¼ 8), and show that b-oxidation defects can be detected in our experiments. Overall, we propose an original laboratory test to investigate FAO in human skeletal muscle and to screen for FAO disorders in myopathies and cardiomyopathies in human.

Summary Increased mitochondrial biogenesis by activation of PPAR- or AMPK/PGC-1α-dependent homeostatic pathways has been proposed as a treatment for mitochondrial disease. We tested this hypothesis on three recombinant mouse models characterized by defective cytochrome c-oxidase (COX) activity: a knockout (KO) mouse for Surf1, a knockout/knockin mouse for Sco2, and a muscle-restricted KO mouse for Cox15. First, we demonstrated that double-recombinant animals overexpressing PGC-1α in skeletal muscle on a Surf1 KO background showed robust induction of mitochondrial biogenesis and increase of mitochondrial respiratory chain activities, including COX. No such effect was obtained by treating both Surf1−/− and Cox15−/− mice with the pan-PPAR agonist bezafibrate, which instead showed adverse effects in either model. Contrariwise, treatment with the AMPK agonist AICAR led to partial correction of COX deficiency in all three models, and, importantly, significant motor improvement up to normal in the Sco2KO/KI mouse. These results open new perspectives for therapy of mitochondrial disease., Highlights ► AICAR partially corrects COX deficiency in three mouse models ► Bezafibrate, a pan-PPAR activator, failed to correct COX deficiency in vivo ► Motor performance improves under AICAR in one mouse model ► Activation of the AMPK/Pgc1α axis can correct OXPHOS defects in vivo

The alpha isoform of peroxisome proliferator-activated receptor (PPARalpha), which is highly expressed in the kidney, can stimulate the expression of genes that are involved in fatty acid catabolism and therefore might be involved in the control of renal fatty acid beta-oxidation. PPARalpha expression and its regulation in the immature kidney are not well documented. This study delineated the developmental pattern of PPARalpha expression in the rat kidney cortex and the medulla between postnatal days 10 and 30 and investigated the role of glucocorticoids in regulating PPARalpha expression. In the cortex, PPARalpha mRNA and protein increased 2- and 1.8-fold, respectively, from 10 to 21 d and then decreased 1.5- and 2.4-fold from 21 to 30 d. In the medulla, PPARalpha mRNA and protein increased continuously 3.3- and 2.4-fold, respectively. It is shown here that acute treatment by dexamethasone of 10-d-old rats precociously induced a 4- to 6-fold increase in PPARalpha mRNA and a 1.8-fold increase in protein within 6 h in each part of the kidney. Chronic injection of dexamethasone for 3 d also increased PPARalpha mRNA 3.8- and 2.2-fold in the cortex and the medulla, respectively, with a 1.5- and 2-fold increase in protein. Furthermore, adrenalectomy prevented the increases in PPARalpha mRNA and protein in both the cortex and the medulla between postnatal days 16 and 21, and these could be restored by dexamethasone treatment. Finally, with the use of an established renal cell line, it was shown that glucocorticoids stimulate gene expression of PPARalpha and of medium chain acyl-CoA dehydrogenase (MCAD, a PPARalpha target gene) 2- to 4-fold and 1.5-fold, respectively, and that addition of fatty acids in the culture media led to a 2.2-fold increase in MCAD mRNA. Altogether, these results demonstrated that glucocorticoids are potent regulators of PPARalpha development in the immature kidney and that these hormones act in concert with fatty acids to regulate MCAD gene expression in renal cells.

The expression of the biogenic amine degrading enzyme monoamine oxidases-A and -B depends on several factors including regional distribution, development and hormonal environment. In the present study, we investigated the expression of monoamine oxidases in developing kidney and their regulation by dexamethasone treatment. Immunoblots and enzyme assays, performed using [14C]5-hydroxytriptamine and [14C]beta-phenylethylamine as substrates for monoamine oxidases-A and -B, respectively, showed that monoamine oxidase-A is the isoenzyme largely predominant in 9-day-old rats renal cortex. Experiments performed in 5-week-old rats showed an increase in monoamine oxidase-B activity and a decrease in monoamine oxidase-A activity and substrate affinity. The changes of monoamine oxidase-A activity and affinity were mimicked by dexamethasone treatment (0.60 mg/kg body weight injected subcutaneously three times at intervals of 24 h) of 9-day-old rats. In contrast, dexamethasone administration induced a modification of monoamine oxidase-B activity opposite to that found between 9-day- and 5-week-old rats. Dexamethasone treatment did not modify immunoreactivity and mRNA corresponding to monoamine oxidases-A and -B indicating that changes of enzyme activities were unrelated to regulation of protein synthesis and mRNA turnover. These results show that monoamine oxidases-A and -B are differently expressed in developing renal cortex and are regulated by dexamethasone treatment.

Regulation of genes involved in fatty acid (FA) utilization in heart and liver of weanling rats was investigated in response to variations in dietary lipid content and to changes in intracellular FA homeostasis induced by etomoxir, a blocker of FA import into mitochondria. Northern-blot analyses were performed using cDNA probes specific for FA transport protein, a cell membrane FA transporter; long-chain- and medium-chain acyl-CoA dehydrogenases, which catalyze the first step of mitochondrial FA beta-oxidation; and acyl-CoA oxidase, a peroxisomal FA beta-oxidation marker. High-fat feeding from postnatal d 21 to 28 resulted in a coordinate increase (58 to 136%) in mRNA abundance of all genes in heart. In liver, diet-induced changes in mitochondrial and peroxisomal beta-oxidation enzyme mRNAs (from 52 to 79%) occurred with no change in FA transport protein gene expression. In both tissues, the increases in mRNA levels went together with parallel increases in enzyme activity. Changes in FA homeostasis resulting from etomoxir administration led to a marked stimulation (76 to 180%) in cardiac expression of all genes together with parallel increases in enzyme activities. In the liver, in contrast, etomoxir stimulated the expression of acyl-CoA oxidase gene only. Feeding rats a low-fat diet containing 0.5% clofibrate, a ligand of peroxisome proliferator-activated receptor alpha, resulted in similar inductions of beta-oxidation enzyme genes in both tissues, whereas up-regulation of FA transport protein gene was restricted to heart. Altogether, these data suggest that changes in FA homeostasis in immature organs resulting either from high-fat diet or beta-oxidation blockade can efficiently be transduced to the level of gene expression, resulting in tissue-specific adaptations in various FA-using enzymes and proteins.

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid beta-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the alpha-isoform of peroxisome proliferator-activated receptor (PPARalpha) in mediating beta-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16 to 21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of beta-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARalpha agonist, demonstrated that PPARalpha can mediate regulation of cortical beta-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal beta-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARalpha-mediated mechanisms.

International audience; Exposure to fibrates leads to normalization of fatty acid oxidation (FAO) in fibroblasts from patients with myopathic forms of CPT2 deficiency or VLCAD deficiency. Correction of FAO is related to a drug-induced increase of residual enzyme activity, and this could provide a new treatment strategy for these disorders.

Background During the development of heart failure (HF), the chief myocardial energy substrate switches from fatty acids to glucose. This metabolic switch, which recapitulates fetal cardiac energy substrate preferences, is thought to maintain aerobic energetic balance. The regulatory mechanisms involved in this metabolic response are unknown. Methods and Results To characterize the expression of genes involved in mitochondrial fatty acid β-oxidation (FAO) in the failing heart, levels of mRNA encoding enzymes that catalyze the first and third steps of the FAO cycle were delineated in the left ventricles (LVs) of human cardiac transplant recipients. FAO enzyme and mRNA levels were coordinately downregulated (>40%) in failing human LVs compared with controls. The temporal pattern of this alteration in FAO enzyme gene expression was characterized in a rat model of progressive LV hypertrophy (LVH) and HF [SHHF/Mcc- fa cp (SHHF) rat]. FAO enzyme mRNA levels were coordinately downregulated (>70%) during both the LVH and HF stages in the SHHF rats compared with controls. In contrast, the activity and steady-state levels of medium-chain acyl-CoA dehydrogenase, which catalyzes a rate-limiting step in FAO, were not significantly reduced until the HF stage, indicating additional control at the translational or posttranslational levels in the hypertrophied but nonfailing ventricle. Conclusions These findings identify a gene regulatory pathway involved in the control of cardiac energy production during the development of HF.

We report on the loss of mitochondrial nicotinamide adenine dinucleotides in human cultured cells along with cell culture and acidification of the culture medium. This was established both by the direct measurement of the decrease in the mitochondrial NAD content and by the alteration of the oxidative properties of the mitochondria. In situ, this loss could be reversed in less than 2 h by changing the culture medium or by readjusting the pH of the medium at physiological pH values. By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD+-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD+ into the mitochondrial matrix. These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. They also point to the possible confusion between a loss of mitochondrial NAD and a defect of respiratory chain complex I in the context of screening procedures for respiratory chain disorder in human.

Mitochondrial fatty acid β-oxidation plays a major role in providing the ATP required for reabsorptive processes in the adult rat kidney. However, the molecular mechanisms and signals involved in induction of the enzymes of fatty acid oxidation during development in this and other organs are unknown. We therefore studied the changes in the steady-state levels of mRNA encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyses the initial step in mitochondrial fatty acid β-oxidation, in the rat kidney cortex and medulla between postnatal days 10 and 30. Furthermore, we investigated whether the expression of MCAD and of mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid cycle, might be co-ordinately regulated by circulating glucocorticoids in the immature kidney during development. In the cortex, the levels of MCAD mRNA rose 4-fold between day 10 and day 21, and then decreased from day 21 to day 30. In the medulla a postnatal increase in the concentration of MCAD mRNA (8-fold) was observed during the same period. Adrenalectomy prevented the 16–21-day developmental increases in MCAD and mMDH mRNA levels in the cortex and medulla; these could be restored by dexamethasone treatment. A single injection of dexamethasone into 10-day-old rats led to a rise in MCAD and mMDH mRNA levels in the renal cortex due to stimulation of gene transcription, as shown by nuclear run-on assays. Therefore MCAD and mMDH gene expression is tightly regulated at the transcriptional level by developmental changes in circulating glucocorticoid levels. These hormones might thus represent a good candidate as a co-ordinating factor in the expression of nuclear genes encoding mitochondrial enzymes in the kidney during postnatal development.

Ketone bodies represent preferred energy substrates in the adult rat proximal tubule. They are abundant in the plasma of suckling rats and might represent an important oxidative substrate for the immature proximal tubule. The postnatal development of two enzymes involved in ketone body oxidation pathway, 3-ketoacid-CoA transferase and acetoacetyl-CoA thiolase, and of citrate synthase and carnitine acetyltransferase was studied in microdissected rat proximal convoluted tubule (PCT) at 1, 8, 16, and 21 days after birth. The enzyme levels in PCT of juxtamedullary and subcapsular nephrons were compared at 8, 16, and 21 days. A role of thyroid hormones in regulating the development of these enzymes was investigated by studying 8- and 21-day-old pups made hypothyroid by propylthiouracyl (PTU) treatment, as well as 21-day hyperthyroid rats. PTU treatment had no effect on enzyme activities on day 8. In contrast, the activity of all mitochondrial enzymes, except acetoacetyl-CoA thiolase, was significantly decreased in 21-day-old hypothyroid pups. In hypothyroid animals, the normal development of 3-ketoacid-CoA transferase, citrate synthase, and carnitine acetyltransferase could be restored after treatment by triiodothyronine (T3). In addition, one single injection of T3 to 8-day-old control pups induced a precocious rise in the activity of 3-ketoacid-CoA transferase, citrate synthase, and carnitine acetyltransferase in juxtamedullary PCT and in the activity of citrate synthase and carnitine acetyltransferase in subcapsular PCT. Altogether, these results point out the importance of the postnatal physiological rise in T3 in triggering the development of some mitochondrial oxidative enzymes in the PCT.

Background Pulmonary surfactant, which is synthesized by alveolar type II cells (ATII cells) almost exclusively, plays a major role in maintaining alveolar homeostasis by reducing surface tension at the fluid-gas interface. Phosphatidylcholine (PC), the main surfactant lipid component, is largely responsible for this surface activity. The effects of halothane on the phospholipid metabolism of the pulmonary surfactant by ATII cells are unknown, even though these cells are exposed directly to volatile anesthetics during anesthesia and even though any alteration in surfactant biosynthesis by anesthetics may have deleterious effects on lung function and thereby facilitate postoperative pulmonary complications. In the current study, the effects of halothane exposure on surfactant synthesis by rat ATII cells in primary culture were investigated. Methods ATII cells were isolated from adult rat lungs and used for the experiments after 24 h in primary culture. The ability of ATII cells to synthesize surfactant was assessed by the incorporation of radioactive precursors in PC. Cytotoxicity was measured by the rate of lactate dehydrogenase release into the culture medium, and the lactate metabolism was taken as an index of glycolytic metabolism. All metabolic measurements were made after 24 h in primary culture. Effects of various halothane concentrations (1, 2, 4, and 8%) exposure for 4 h were studied, as were the effects of 2% halothane for various durations of exposure (2, 4, 8, and 12 h). The reversibility of halothane effects on PC synthesis was assessed after a 2% halothane exposure for 4 h. PC secretion and adenosine triphosphate cellular content were also measured for 4 h exposure at the various halothane concentrations. Results During a 4-h exposure, PC synthesis was reduced by 10, 24, 29 and 36% for 1, 2, 4, and 8% halothane respectively when compared with control values. At 2% halothane concentration, the observed decreases in PC synthesis were 12, 24, 31 and 34% for 2, 4, 8, and 12 h exposure, respectively. The inhibitory effect of halothane was completely reversed 2 h after the end of exposure. PC secretion was unaffected by increasing halothane concentrations during a 4-h exposure. Halothane did not produce cell damage except for the longest exposure durations (8 and 12 h) at 2% vapor concentration. Whatever the exposure conditions, lactate production by ATII cells exposed to halothane was greater than production by unexposed cells. Conclusions These results indicate that halothane decreases the biosynthesis of pulmonary surfactant by ATII cells in primary culture and alters the high energy phosphate metabolism of these cells.

We have previously shown that the activity of several mitochondrial oxidative enzymes increased greatly in the medullary thick ascending limb of Henle's loop (MTAL) of developing rat kidney between 16 days after birth and the adult stage. These changes were triggered by the postnatal rise in circulating glucocorticoids. To determine whether these increases are related to a mitochondrial biogenesis we have studied the changes in mitochondrial density of MTAL cells and mitochondrial DNA content in the inner stripe of the outer medulla during the postnatal period. The activities of respiratory chain (RC) complexes II and IV, located in the inner mitochondrial membrane (IMM), were also assayed, and developmental changes in the ultrastructure of the IMM were quantified. Quantitative electron-microscopic data showed a doubling in mitochondrial density from day 16 to the adult, accompanied by twofold increase in mitochondrial DNA, as determined by slot-blot experiments. The surface density of IMM increased by 77%, and there was a concomitant large rise in the activity of both RC enzymes. A possible role of glucocorticoids on the regulation of mitochondrial biogenesis was examined in similar experiments performed in adrenalectomized rat pups. The data demonstrated that glucocorticoids are essential for the rise in RC enzymes and the development of IMM but do not regulate postnatal changes in mitochondrial density and mitochondrial DNA content. Finally, ontogenic changes in oxidative capacities of MTAL cells could be a critical factor in the development of kidney urine concentrating mechanisms in weanling rats.

The postnatal development of mitochondrial ATP-producing pathways and Na-K-adenosinetriphosphatase (ATPase) in the rat medullary thick ascending limb of Henle (MTAL) was studied by measuring the activities of 3-ketoacid-CoA transferase, fumarase, citrate synthase, and Na-K-ATPase in microdissected MTAL of 16, 21, and 30-day-old pups and in adults. The role of adrenal steroids in the development of these four markers was also investigated by studying 21-day-old rats adrenalectomized on day 16 and given dexamethasone or aldosterone or NaCl injections from day 16 to day 21. There were large and correlated increases in the activities of the oxidative enzymes in the MTAL of control rat kidneys between 16 and 30 days after birth; Na-K-ATPase activity in the MTAL also greatly increased during the same period. Adrenalectomy completely prevented the developmental increases in MTAL oxidative enzymes and Na-K-ATPase; dexamethasone restored the development of all four enzymes, whereas aldosterone had no effect. We conclude that the postnatal maturation of Na+ reabsorption functions in MTAL cells involves coordinated increases in the capacity to produce ATP by oxidative metabolism and in Na-K-ATPase activity. This maturation process is probably triggered by the rise in circulating glucocorticoids that occurs during the weaning period.

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ß-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme–FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer–monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach.

During the 1 st h of extrauterine life, the adenine nucleotide content of the rat kidney is modified: the ATP level increases (+30%) while ADP and AMP are lowered (––30 and ––50%, respectively). This leads to a high value of energy charge in the newborn kidney (0.89 vs. 0.80 in the fetus). It was possible to obtain in utero a similar modification of ATP, ADP, AMP concentrations by injections to the fetuses of cAMP, dibutyryl cAMP, or isoprenaline. Conversely, the postnatal changes in adenine nucleotide content could be prevented by administration to the fetus, just before birth, of β- or β1-adrenoreceptor antagonists. Therefore the rise of kidney energy charge following parturition appears to be under hormonal control. Glucagon had no effect on kidney adenine nucleotide content. It is strongly suggested that the catecholamines released at the time of parturition are the triggering factor of this evolution.

The oxygen-consumption rates and the activities of fumarase and beta-hydroxyacyl-CoA dehydrogenase were compared in mitochondria isolated from fetal- and neonatal-rat kidney. Whole-organ ATP, phosphocreatine and creatine contents were determined in parallel. Kidney mitochondrial respiratory rates in the presence of succinate, glutamate/malate and palmitoyl-L-carnitine increased between 21 days post coitum and 1 day post partum, together with activities of oxidative enzymes. However, this postnatal maturation of oxidative metabolism was not yet initiated in mitochondria isolated from kidney 1 h post partum. An increase in ATP and phosphocreatine was observed immediately after delivery; newborn-rat kidney ATP content then remained high, whereas phosphocreatine reserves decreased considerably between 6 h and 1 day post partum. It is concluded that the increase in high-energy phosphate compounds observed at birth is not initially related to an activation of oxidative phosphorylation, and probably involves a transient stimulation of anaerobic glycolysis, while a progressive mitochondrial maturation takes place in the rat kidney during the first day of newborn life.

Change in energy reserves in different segments of the nephron during brief ischemia. Rat kidneys were made ischemic for 5 to 120 seconds. Segments of individual nephrons were dissected from freeze dried sections and analyzed for ATP, phosphocreatine, glycogen, glucose, glucose-6-phosphate, lactate and creatine kinase. ATP fell most rapidly in proximal convoluted and straight tubules (PCT, PST) and distal convoluted tubules (DCT), and most slowly in glomerulus and papilla. Phosphocreatine levels ranged fivefold and was highest in DCT, where it approached that of brain. Creatine kinase ranged 100-fold with lowest level in PCT, where the ischemic fall in phosphocreatine was so slow as to suggest a function other than that of an energy reserve. Glycogen varied tenfold from modest levels in distal segments to very low levels in PST, and was not used rapidly in any segment. Glucose consumption and lactate production were most rapid in distal portions. High–energy phosphate consumption for the first 7.5 seconds of ischemia, calculated from these data, indicates roughly–equal energy metabolism in proximal and distal segments, with lower levels in papilla, and especially in glomerulus. The absolute values suggest that the in vivo metabolic rate of the nephron continued almost unabated for 5 or 10 seconds of ischemia.