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Patients with autism spectrum disorder (ASD) may have an increase in blood acyl-carnitine (AC) concentrations indicating a mitochondrial fatty acid β-oxidation (mtFAO) impairment. However, there are no data on systematic mtFAO analyses in ASD. We analyzed tritiated palmitate oxidation rates in fibroblasts from patients with ASD before and after resveratrol (RSV) treatment, according to methods used for the diagnosis of congenital defects in mtFAO. ASD participants (N = 10, 60%, male, mean age (SD) 7.4 (3.2) years) were divided in two age-equivalent groups based on the presence (N = 5) or absence (N = 5) of elevated blood AC levels. In addition, electron transport chain (ETC) activity in fibroblasts and muscle biopsies and clinical characteristics were compared between the ASD groups. Baseline fibroblast mtFAO was not significantly different in patients in comparison with control values. However, ASD patients with elevated AC exhibited significantly decreased mtFAO rates, muscle ETC complex II activity, and fibroblast ETC Complex II/III activity (p &lt, 0.05), compared with patients without an AC signature. RSV significantly increased the mtFAO activity in all study groups (p = 0.001). The highest mtFAO changes in response to RSV were observed in fibroblasts from patients with more severe symptoms on the Social Responsiveness Scale total (p = 0.001) and Awareness, Cognition, Communication and Motivation subscales (all p &lt, 0.01). These findings suggested recognition of an ASD patient subset characterized by an impaired mtFAO flux associated with abnormal blood AC. The study elucidated that RSV significantly increased fibroblast mtFAO irrespective of plasma AC status, and the highest changes to RSV effects on mtFAO were observed in the more severely affected patients.

International audience; Inherited fatty acid oxidation diseases in their mild forms often present as metabolic myopathies. Carnitine Palmitoyl Transferase 2 (CPT2) deficiency, one such prototypical disorder is associated with compromised myotube differentiation. Here, we show that CPT2-deficient myotubes exhibit defects in focal adhesions and redox balance, exemplified by increased SOD2 expression. We document unprecedented alterations in the cellular prion protein PrPC, which directly arise from the failure in CPT2 enzymatic activity. We also demonstrate that the loss of PrPC function in normal myotubes recapitulates the defects in focal adhesion, redox balance and differentiation hallmarks monitored in CPT2-deficient cells. These results are further corroborated by studies performed in muscles from Prnp-/- mice. Altogether, our results unveil a molecular scenario, whereby PrPC dysfunction governed by faulty CPT2 activity may drive aberrant focal adhesion turnover and hinder proper myotube differentiation. Our study adds a novel facet to the involvement of PrPC in diverse physiopathological situations.

International audience; Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis but its contribution to macrophage functions and host defense remains to be delineated. Here we showed that lipopolysaccharide (LPS) in combination with IFNγ, inhibits PINK1-dependent mitophagy in macrophages through a STAT1-dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggers classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture, CLP), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation which favored bactericidal clearance and lead to a better survival. Reciprocally, mitochondrial uncouplers, that promote mitophagy, reverse LPS/IFNγ-mediated activation of macrophages and lead to immuno-paralysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy is inhibited in blood monocytes of patients with sepsis as compared to non-septic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis.

International audience; Reactive oxygen species (ROS) modify proteins and lipids leading to deleterious outcomes. Thus, maintaining their homeostatic levels is vital. This study highlights the endogenous role of LXRs (LXRα and β) in the regulation of oxidative stress in peripheral nerves. We report that the genetic ablation of both LXR isoforms in mice (LXRdKO) provokes significant locomotor defects correlated with enhanced anion superoxide production, lipid oxidization and protein carbonylation in the sciatic nerves despite the activation of Nrf2-dependant antioxidant response. Interestingly, the reactive oxygen species scavenger N-acetylcysteine counteracts behavioral, electrophysical, ultrastructural and biochemical alterations in LXRdKO mice. Furthermore, Schwann cells in culture pretreated with LXR agonist, TO901317, exhibit improved defenses against oxidative stress generated by tert-butyl hydroperoxide, implying that LXRs play an important role in maintaining the redox homeostasis in the peripheral nervous system. Thus, LXR activation could be a promising strategy to protect from alteration of peripheral myelin resulting from a disturbance of redox homeostasis in Schwann cell. Liver X Receptors (LXRα and LXRβ) belong to the nuclear receptor superfamily of ligand-activated transcription factors. They regulate target gene expression by binding to specific responsive elements and are implicated in metabolic processes such as cholesterol turnover, inflammation as well as pathologies such as cancer and neu-rodegenerative diseases 1. Natural ligands of LXRs are oxysterols (i.e. 24(S)-hydroxycholesterol (24(S)-OH) or 25-hydroxycholesterol (25-OH)), produced either through auto-oxidation or enzymatic oxidation of cholesterol. Synthetic ligands of LXR, like TO901317, have also been discovered and are known to be potent activators of the LXR pathway. Previous studies have shown that mice where both LXR isoforms (LXRα and LXRβ) are deleted (LXR double KO or LXRdKO) exhibit altered lipid homeostasis in the brain resulting in neuronal loss, astrocytic proliferation , disorganized myelin sheaths and lipid accumulation in specific brain regions that participates in locomo-tor defects highlighted in these animals 2,3. We also observed that LXRdKO mice have thinner myelin sheaths surrounding axons of the sciatic nerve 4,5. Importantly, LXR inhibition enhanced myelin gene transcripts but decreased the amount of myelin proteins, suggesting post-translational modifications, detrimental for peripheral myelin integrity. Oxidative stress has been recently shown to alter the structure of myelin proteins in several diabetic peripheral neuropathies 6. In particular, PMP22 misfolding and aggregation provokes demyelination and nerve conduction velocity reduction. In line with this, our team recently showed that a burst of oxidative stress induced by Paraquat provokes a dramatic alteration of myelin structure in the sciatic nerves 7. Indeed, because of their high reactivity, reactive oxygen species (ROS) modify the structure and therefore the physiological functions of proteins and lipids. Thus, maintaining normal cellular ROS levels is essential. The excessive production of ROS or the decrease of antioxidant defenses is rather a hallmark characteristic in the pathogenesis of diseases such as diabetes, athero-sclerosis and neurodegeneration 8,9. Hence, compounds that exhibit anti-oxidative effects, triggering the intracel-lular cascade of protective pathways, may offer a promising strategy for therapeutic applications 10 .

International audience; Spinal Muscular Atrophy (SMA), an autosomal recessive neurodegenerative disease characterized by the loss of spinal-cord motor-neurons, is caused by mutations on Survival-of-Motor Neuron (SMN)-1 gene. The expression of SMN2, a SMN1 gene copy, partially compensates for SMN1 disruption due to exon-7 excision in 90% of transcripts subsequently explaining the strong clinical heterogeneity. Several alterations in energy metabolism, like glucose intolerance and hyperlipidemia, have been reported in SMA at both systemic and cellular level, prompting questions about the potential role of energy homeostasis and/or production involvement in disease progression. In this context, we have recently reported the tolerance of mild SMA-like mice (SmnΔ7/Δ7; huSMN2 +/+) to 10 months of low-intensity running or high-intensity swimming exercise programs, respectively involving aerobic and a mix aerobic/anaerobic muscular metabolic pathways. Here, we investigated whether those exercise-induced benefits were associated with an improvement in metabolic status in mild SMA-like mice. We showed that untrained SMA-like mice exhibited a dysregulation of lipid metabolism with an enhancement of lipogenesis and adipocyte deposits when compared to control mice. Moreover, they displayed a high oxygen consumption and energy expenditure through β-oxidation increase yet for the same levels of spontaneous activity. Interestingly, both exercises significantly improved lipid metabolism and glucose homeostasis in SMA-like mice, and enhanced oxygen consumption efficiency with the maintenance of a high oxygen consumption for higher levels of spontaneous activity. Surprisingly, more significant effects were obtained with the high-intensity swimming protocol with the maintenance of high lipid oxidation. Finally, when combining electron microscopy, respiratory chain complexes expression and enzymatic activity measurements in muscle mitochondria, we found that (1) a muscle-specific decreased in enzymatic activity of respiratory chain I, II, and IV complexes for equal amount of mitochondria and complexes expression and (2) a significant decline in mitochondrial maximal oxygen consumption, were reduced by both exercise programs. Most of the beneficial effects were obtained with the high-intensity swimming protocol. Taking together, our data support the hypothesis that active physical exercise, including high-intensity protocols, induces metabolic adaptations at both systemic and cellular levels, providing further evidence for its use in association with SMN-overexpressing therapies, in the long-term care of SMA patients.

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondria] function, and more recently it was shown to restore fatty acid beta-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1 alpha, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the alpha subunit of the stimulatory G protein (Gs alpha), which regulates the cellular levels of cAMP through adenylyl cyclase.We conclude that in fibroblasts RSV stimulates the PGC-1 alpha and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial beta-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

International audience; Carnitine palmitoyl transferase 2 (CPT2) deficiency is one of the most common inherited fatty acid oxidation (FAO) defects and represents a prototypical mitochondrial metabolic myopathy. Recent studies have suggested a pivotal role of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle plasticity and mitochondrial homeostasis. Thus, we tested the potential of GSK773, a novel direct AMPK activator, to improve or correct FAO capacities in muscle cells from patients harboring various mutations. We used controls' and patients' myotubes and studied the parameters of FAO metabolism, of mitochondrial quantity and quality and of differentiation. We found that AMPK is constitutively activated in patients' myotubes, which exhibit both reduced FAO and impaired differentiation. GSK773 improves or corrects several metabolic hallmarks of CPT2 deficiency (deficient FAO flux and C16-acylcarnitine accumulation) by upregulating the expression of CPT2 protein. Beneficial effects of GSK773 are also likely due to stimulation of mitochondrial biogenesis and induction of mitochondrial fusion, by decreasing dynamin-related protein 1 and increasing mitofusin 2. GSK773 also induces a shift in myosin heavy chain isoforms toward the slow oxidative type and, therefore, fully corrects the differentiation process. We establish, through small interfering RNA knockdowns and pharmacological approaches, that these GSK773 effects are mediated through peroxisome proliferator-activated receptor gamma co-activator 1-alpha, reactive oxygen species and p38 mitogen-activated protein kinase, all key players of skeletal muscle plasticity. GSK773 recapitulates several important features of skeletal muscle adaptation to exercise. The results show that AMPK activation by GSK773 evokes the slow, oxidative myogenic program and triggers beneficial phenotypic adaptations in FAO-deficient myotubes. Thus, GSK773 might have therapeutic potential for correction of CPT2 deficiency.

International audience; The pathophysiological mechanisms underlying Complex I (CI) deficiencies are understood only partially which severely limits the treatment of this common, devastating, mitochondrial disorder. Recently, we have shown that resveratrol (RSV), a natural polyphenol, has beneficial effects on CI deficiency of nuclear origin. Here, we demonstrate that RSV is able to correct the biochemical defect in oxygen consumption in five of thirteen CI-deficient patient cell lines. Other beneficial effects of RSV include a decrease of total intracellular ROS and the up-regulation of the expression of mitochondrial superoxide dismutase (SOD2) protein, a key antioxidant defense enzyme. The molecular mechanisms leading to the up-regulation of SOD2 protein expression by RSV require the estrogen receptor (ER) and the estrogen-related receptor alpha (ERRα). Although RSV increases the level of SOD2 protein in patients' fibroblasts, the enzyme activity is not increased, in contrast to normal fibroblasts. This led us to hypothesize that SOD2 enzyme activity is regulated post-translationally. This regulation involves SIRT3, a mitochondrial NAD þ-dependent deacetylase and is critically dependent on NAD þ levels. Taken together, our data show that the metabolic effects of RSV combined with its antioxidant capacities makes RSV particularly interesting as a candidate molecule for the therapy of CI deficiencies.

International audience; Pyruvate carboxylase (PC) is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA) cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A). Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV) defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1) and type B (patient 2). Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

International audience; SCOPE:High-fat diet (HFD) increases visceral adipose tissue (AT). Our aim was to evaluate whether citrulline (CIT) affected nonesterified fatty acid (NEFA) metabolism in AT from HFD-fed rats.METHODS AND RESULTS:Rats were fed for 8 weeks with either a control diet (CD) or HFD. Retroperitoneal AT explants were exposed to 2.5 mmol/L CIT for 24 h. We analyzed lipolysis, beta-oxidation, glyceroneogenesis, and the expression of the key associated enzymes. CIT doubled NEFA release selectively in HFD AT. Phosphorylation of hormone-sensitive lipase was upregulated 50 and 100% by CIT in CD and HFD AT, respectively. Under CIT, beta-oxidation increased similarly whatever the diet, whereas glyceroneogenesis, which permits NEFA re-esterification, was downregulated 50 and 80% in CD and HFD AT, respectively. In the latter, the important decrease in re-esterification probably explains the rise of NEFA release. A pretreatment with the nitric oxide synthase inhibitor N ω-nitro-l-arginine methyl ester abolished CIT effects.CONCLUSION:These results demonstrate direct lipolytic and antiglyceroneogenic effects of CIT on CD and HFD AT. The selective CIT-mediated NEFA release from HFD AT was probably the consequence of the drastic decrease in glyceroneogenesis and nitric oxide was a mediator of CIT effects. These results provide evidence for a direct action of CIT on AT to reduce overweight.

International audience; Background: Inborn enzyme defects of mitochondrial fatty acid beta-oxidation (FAO) form a large group of genetic disorders associated to variable clinical presentations ranging from life-threatening pediatric manifestations up to milder late onset phenotypes, including myopathy. Very few candidate drugs have been identified in this group of disorders. Resveratrol (RSV) is a natural polyphenol with anti-oxidant and anti-inflammatory effects, recently shown to have beneficial metabolic properties in mice models. Our study explores its possible effects on FAO and mitochondrial energy metabolism in human cells, which are still very little documented.Methods: Using cells from controls and from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency we characterized the metabolic effects of RSV, RSV metabolites, and other stilbenes. We also focused on analysis of RSV uptake, and on the effects of low RSV concentrations, considering the limited bioavailability of RSV in vivo.Results: Time course of RSV accumulation in fibroblasts over 48 h of treatment were consistent with the resulting stimulation or correction of FAO capacities. At 48 h, half maximal and maximal FAO stimulations were respectively achieved for 37,5 microM (EC50) and 75 microM RSV, but we found that serum content of culture medium negatively modulated RSV uptake and FAO induction. Indeed, decreasing serum from 12% to 3% led to shift EC50 from 37,5 to 13 microM, and a 2.6-3.6-fold FAO stimulation was reached with 20 microM RSV at 3% serum, that was absent at 12% serum. Two other stilbenes often found associated with RSV, i.e. cis- RSV and piceid, also triggered significant FAO up-regulation. Resveratrol glucuro- or sulfo- conjugates had modest or no effects. In contrast, dihydro-RSV, one of the most abundant circulating RSV metabolites in human significantly stimulated FAO (1.3-2.3-fold).Conclusions: This study provides the first compared data on mitochondrial effects of resveratrol, its metabolites, and other natural compounds of the stilbene family in human cells. The results clearly indicate that several of these compounds can improve mitochondrial FAO capacities in human FAO-deficient cells.

International audience; Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 μM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.

International audience; Carnitine palmitoyl transferase 2 (CPT2) and very-long-chain Acyl-CoA dehydrogenase (VLCAD) deficiencies are among the most common inborn mitochondrial fatty acid b-oxidation (FAO) disorders. Despite advances in their clinical and molecular characterizations, few therapeutic approaches exist for these diseases. Resveratrol (RSV) is a natural polyphenol extensively studied for its potential health benefits. Indeed, it is presently thought that RSV could delay the onset of some cancers, and have protective effects against common aging disorders such as type II diabetes, cardiovascular or neurodegenerative diseases. Here, we show that exposure to RSV induces a dose-and time-dependant increase in FAO flux in human fibroblasts, and can restore normal FAO capacities in a panel of patients' fibroblasts with the mild forms (harboring various genotypes) of CPT2 or VLCAD deficiency. The correction of FAO flux correlated with a marked increase in mutant CPT2 or VLCAD protein level, in cells treated by RSV. Inhibition of sirtuin 1 (SIRT1) by Sirtinol and the use of peroxisome proliferator-activated receptor gamma co-activator-1-alpha (PGC-1a) small interfering RNAs demonstrate that the RSV-induced stimulation of FAO requires the presence of PGC-1a and SIRT1. These results show, for the first time, that RSV markedly induces mitochondrial FAO capacities in human fibroblasts, and provides the initial proof-of-concept that RSV might be efficient for correction of inherited FAO disorders.

International audience; Carnitine palmitoyltransferase 2 (CPT2) deficiency is a rare mitochondrial fatty acid oxidation (FAO) disorder characterized by myalgia, exercise intolerance, and rhabdomyolysis. We evaluate the efficacy of bezafibrate (BZ), a hypolipidemic drug, as a treatment for this form of CPT2 deficiency. A pilot trial was conducted with BZ in six patients for 6 months. There was a follow-up period of 3 years. The oxidation rates of the long-chain fatty acid derivative palmitoyl-CoA, measured in the mitochondria of the patients' muscles, were markedly lower than normal before treatment and increased significantly (+39 to +206%; P = 0.028) in all patients after BZ treatment. The evaluation of the therapeutic effects by the patients themselves (using the Short Form Health Survey (SF-36)), as well as by the physicians, indicated an improvement in the condition of the patients; there was an increase in physical activity and a decline in muscular pain. The results suggest that BZ has a therapeutic effect in the muscular form of CPT2 deficiency.

To the Editor: Carnitine palmitoyltransferase II (CPT2) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation. The most common form of this disorder is characteriz...

International audience; Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid b-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in b-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 mM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.

International audience; Type 2 carnitine palmitoyl transferase (CPT2) is involved in the transfer of long-chain fatty acid into the mitochondria. CPT2-deficient patients carry gene mutations associated with different clinical presentations, correlating with various levels of fatty acid oxidation (FAO) and residual CPT2 enzyme activity. We tested the hypothesis that pharmacological stimulation of peroxisome proliferator-activated receptors (PPAR) can stimulate FAO in CPT2-deficient muscle cells. Accordingly, we show that a 48-h treatment of CPT2-deficient myoblasts by bezafibrate restored FAO in patient cells. Specific agonists of PPARdelta (GWdelta 0742), and, to a lower extent, PPARalpha (GWalpha 7647) also stimulated FAO in control myoblasts. However, when tested in CPT2-deficient myoblasts, only the delta-agonist was able to restore FAO, whereas the alpha-agonist had no effect. GWdelta 0742 increased CPT2 mRNA levels, whereas no change in CPT2 transcripts was found in response to GWalpha 7647. Bezafibrate and GWdelta 0742 increased residual CPT2 activity and normalized long-chain acylcarnitine production by deficient cells. Finally, CPT1-B mRNA was also stimulated after PPAR agonist treatment, and this likely takes part in drug-induced increase of FAO in control muscle cells. In conclusion, this study clearly suggests that PPARs could be therapeutic targets for correction of inborn beta-oxidation defects in human muscle. Furthermore, these data also illustrate a selective control of beta-oxidation enzyme gene expression by PPARdelta, with no contribution of PPARalpha.

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.

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.