Fatima Djouadi

Jean Bastin
email: jean.bastin@inserm.fr

Species differences in the effects of bezafibrate as a potential treatment of mitochondrial disorders

Dear Editor, Genetic defects of mitochondrial respiratory chain form an expanding family of rare diseases, whose number and global incidence increases constantly whereas treatment options remain extremely limited. In line with recent literature data [START_REF] Bastin | Activation of peroxisome proliferator-activated receptor pathway stimulates the mitochondrial respiratory chain and can correct deficiencies in patients' cells lacking its components[END_REF] suggesting a potential of bezafibrate for correction of respiratory chain (RC) defects in human fibroblasts, Viscomi et al recently published in Cell Metabolism the results of in vivo experiments aimed at evaluating the effects of bezafibrate in RC deficient knockout mice [START_REF] Viscomi | In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1alpha Axis[END_REF]. The conclusions from this study appeared in marked contrast with those drawn from in vitro studies in patient cells, and apparently casts doubt on therapeutic properties of bezafibrate in RC deficiencies. However, we consider that limitations in the study design could explain the apparent inefficacy and toxic effects of bezafibrate reported by the authors. Furthermore, based on clinical data obtained in individuals treated with bezafibrate, we present data showing that this drug can stimulate the respiratory chain function in the human skeletal muscle.

One of the most questionable points in the study of Viscomi et al is the bezafibrate dosage tested in the knockout mice (0.5% drug added to standard diet for one month), for several reasons. At first, when using this diet, it is easy to calculate that the daily drug supply is in considerable excess compared to the pharmacological dose used in humans. Indeed, assuming a mouse body weight of 25-30 grams and a 4 grams/day food intake, 0.5% bezafibrate in chow is equivalent to 666-800 mg/kg/day bezafibrate, i.e. represents up to 80fold the dose used for the treatment of dyslipidemia (10 mg/kg/day). The second and main concern is the known toxicity and carcinogenic potential of such high doses of fibrate in rodents, established in the early 1980's. Indeed, it is known that a two-fold increase in liver weight is already observed in mice after 1-week on a diet containing 0.5% bezafibrate, likely due to PPAR-alpha mediated induction of genes involved in hepatocyte proliferation (cyclin D1, CDK4, and c-Myc), whereas mice kept on this regimen will develop hepatocarcinoma in the long term [START_REF] Hays | Role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in bezafibrateinduced hepatocarcinogenesis and cholestasis[END_REF]. Importantly, recent studies also show that clinically-relevant doses of bezafibrate elicit triglyceride-lowering effects in mice, and no toxic effects [START_REF] Nakajima | Bezafibrate at clinically relevant doses decreases serum/liver triglycerides via down-regulation of sterol regulatory element-binding protein-1c in mice: a novel peroxisome proliferator-activated receptor alpha-independent mechanism[END_REF].

Taking into account these literature data, there is no rationale to use 0.5% bezafibrate in diet when investigating pharmacological properties of this drug. Furthermore, it appears likely that liver hepatomegaly reported both in treated Surf 1-/-and wild-type animals reflect a classical toxic response to high doses of bezafibrate. PPAR agonists at high doses can also induce muscle damages (myofibril degeneration and inflammatory cell infiltration).

Accordingly, worsening of muscle damages in ACTA-Cox15-/-mice treated by bezafibrate, could also be ascribed to toxic effects of bezafibrate overdosage. Under these conditions, conclusions on the therapeutic potential of bezafibrate in RC-deficient mouse models cannot be drawn, and extrapolation to the treatment of RC-deficient patients appears irrelevant .

Importantly, the hepatotoxicity and carcinogenic activity of fibrates are clearly rodentspecific. Indeed, it has long been known that humans are resistant to the development of hepatocarcinoma after chronic exposure to fibrates, and, large-scale studies performed since the 1980's consistently established that bezafibrate is a safe drug, with limited side effects (Tenenbaum, A et al, 2005 cited in Bonnefont et al).

Regarding the possible use of this drug in patients with inborn metabolic myopathies, we tested bezafibrate in patients with the myopathic form of Carnitine Palmitoyl Transferase 2 (CPT2) deficiency, one of the commonest inborn mitochondrial fatty acid ß-oxidation defects.

In contrast with the assumption made by Viscomi et al on the basis of their study in mice, this pilot trial did not reveal contraindications in the use of bezafibrate in myopathic patients. On the contrary, CPT2-deficient patients treated by bezafibrate for 6 months at 10 mg/kg/day generally experienced a clear decline in muscular pain and rhabdomyolysis episodes, and less limitation in physical activity. Furthermore, follow-up of these patients for three years indicated stable beneficial effects of the treatment in the long-term, without adverse effects [START_REF] Bonnefont | Long-term follow-up of bezafibrate treatment in patients with the myopathic form of carnitine palmitoyltransferase 2 deficiency[END_REF].

Importantly, as reported here (Supplemental Figure 1), we established in the course of this trial that bezafibrate treatment led to increase the RC capacity in the human skeletal muscle. Indeed, stimulation of RC capacities was reflected by the rise in maximal O2 consumption observed in muscle mitochondria of treated patients. Thus, as shown in Figure 1A, the oxidation rates of pyruvate+malate (a RC complex I substrate) or of succinate (a RC complex II substrate), markedly increased (p=0.028, two-sided Wilcoxon signed-rank test) after 6 months of bezafibrate treatment, in patient muscle mitochondria. Consistent with this, the levels of key respiratory chain proteins i.e. NDUFV1 (Complex I) and COX4 (Complex IV), encoded by nuclear genes, or COX2, a mitochondrial DNA encoded gene, were found strongly increased in the muscle of bezafibrate-treated patients (Figure 1B). Finally, Cytochrome c oxidase (Complex IV, COX) and Citrate Synthase (mitochondrial matrix protein) enzyme activities measured in muscle homogenates significantly increase after bezafibrate treatment (Figures 1C and1D).

Altogether, our in vivo data, as well as in vitro studies performed in patients' cells, reinforce the notion that activation of the PPAR-PGC1 signaling pathway by bezafibrate could be a promising approach for pharmacological correction of partial FAO or RC deficiencies. Clinical trials will be needed to assess the possible beneficial effects of bezafibrate in various RC disorders and the absence of adverse effects. The patients and protocols have been described in detail in reference [START_REF] Bonnefont | Long-term follow-up of bezafibrate treatment in patients with the myopathic form of carnitine palmitoyltransferase 2 deficiency[END_REF]. Briefly, all the patients underwent a first muscle biopsy before receiving bezafibrate and a second biopsy after 6-month of treatment by bezafibrate as three 200-mg tablets per day, which is the regular drug dosage for treatment of hyperlipidemia. These muscle biopsies were primarily used for the measurement of mitochondrial respiratory capacities. When the size of the tissue sample was not limiting, additional parameters were measured to further evaluate the respiratory chain functions.

Figure 1 :

 1 Figure1

( A )

 A Pyruvate plus malate (complex I substrate) and succinate (complex II substrate) oxidation rates in isolated muscle mitochondria from 6 individuals, before (open bars) and after (dark bars) treatment by bezafibrate. O2 consumption rates measured by polarography, are expressed in nmol oxygen consumed per minute per milligram of mitochondrial protein, and represent the average of duplicate measurements. Values obtained in untreated patients were in the normal range (B) Changes in NDUFV1, COX4 and COX2 protein levels in patient muscle before and after treatment by bezafibrate. The protein signals were quantified by computerized analysis. Results of western-blot analysis are expressed in arbitrary units as means ± SD of three different experiments. *p<0.001 versus before treatment (paired t test). (C) Spectrophotometric assay of Cytochrome C oxidase activity in muscle homogenates before and after treatment by bezafibrate. Values are means ± SD of three to five determinations. *p<0.001 versus before treatment (paired t test). (D) Spectrophotometric assay of Citrate Synthase activity in muscle homogenates before and after treatment by bezafibrate. Values are means ± SD of three to six determinations. *p<0.001 versus before treatment (paired t test).


Jean Bastin

Alexandra Lopes-Costa

Fatima Djouadi
email: fatima.djouadi@inserm.fr

Exposure to Resveratrol triggers Pharmacological Correction of Fatty Acid Utilization in Human Fatty Acid Oxidation-deficient Fibroblasts

Carnitine Palmitoyl Transferase 2 (CPT2) and Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiencies are among the most common inborn mitochondrial fatty acid ß-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 resveratrol could delay the onset of some cancers, and have protective effects against common ageing disorders like type II diabetes, cardiovascular, or neurodegenerative diseases. Here, we show that exposure to RSV induces a doseand 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 SIRT1 by Sirtinol and the use of SiRNAs demonstrate that the RSV-induced stimulation of FAO requires the presence of PGC-1α 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.

INTRODUCTION

Mitochondrial fatty acid ß-oxidation (FAO) disorders form a large group of inherited metabolic diseases initially described more that 30 years ago (1, 2). Among these, the Carnitine Palmitoyl Transferase 2 (CPT2) and the Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiencies are considered as relatively common, and they share many characteristics (3,[START_REF] Gregersen | Mutation analysis in mitochondrial fatty acid oxidation defects: Exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship[END_REF]. The CPT2 and VLCAD proteins are both located on the inner mitochondrial membrane. The CPT2 participates in the transfer of long-chain fatty acids into the mitochondria. The long chain acylCoAs produced by CPT2 are the substrates of VLCAD, which catalyzes the initial step of the Lynen helix. A number of tissues, including the heart, the liver or skeletal muscle, are tightly dependent on FAO for energy production, and, accordingly, genetic enzyme defects in this metabolic pathway are generally associated with complex clinical manifestations. In line with this, CPT2 or VLCAD deficiency is associated with several possible phenotypes, which vary in terms of severity, age of onset, and the tissues affected (3,[START_REF] Gregersen | Mutation analysis in mitochondrial fatty acid oxidation defects: Exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship[END_REF]. The most severe form of CPT2 deficiency is characterized by acute liver failure, hypoketotic hypoglycemia, peripheral myopathy and serious cardiac damage, generally fatal during the neonatal period. A severe neonatal phenotype has also been described in VLCAD deficiency, which associates hepatic failure and life-threatening cardiomyopathy. VLCAD deficiency can also manifest itself in infancy by isolated liver failure with episodes of hepatic coma. However, the most common phenotype of both disorders is an adolescent-onset myopathy. Typical manifestations of this metabolic myopathy are muscle stiffness, myalgia, and recurrent attacks of rhabdomyolysis triggered by exercise, fever, or exposure to cold. These muscular forms of CPT2 or VLCAD deficiency are generally considered as relatively mild, as long as life-threatening complications, such as acute renal failure induced by rhabdomyolysis, can be prevented [START_REF] Angelini | Task force guidelines handbook: EFNS guidelines on diagnosis and management of fatty acid mitochondrial disorders[END_REF].

Both disorders have been extensively characterized at the molecular level, revealing a large number of disease-causing mutations in the CPT2 and VLCAD genes (3,[START_REF] Gregersen | Mutation analysis in mitochondrial fatty acid oxidation defects: Exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship[END_REF][START_REF] Corti | Clinical features and new molecular findings in Carnitine Palmitoyltransferase II (CPT II) deficiency[END_REF]. Severely affected patients often harbor null-type mutations (large deletions, truncating, nonsense, etc…), which prevent the production of any protein, thus leading to the collapse cellular FAO capacities and, therefore, accounting for the clinical severity of the associated phenotypes (3,[START_REF] Andresen | Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency[END_REF]. However, and as observed in other FAO defects, the majority of gene variations found in CPT2-or VLCAD-deficient patients are missense mutations [START_REF] Gregersen | Disease mechanisms and protein structures in fatty acid oxidation defects[END_REF]. In the mild presentation characterizing these disorders, patient cell studies have established that gene missense mutations often do not lead to a complete absence of the corresponding protein, but, rather, induce the production of a misfolded, unstable, mutant protein with some residual levels of protein and enzyme activity [START_REF] Andresen | Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency[END_REF][START_REF] Gobin-Limballe | Genetic basis for correction of verylong-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF]10). Despite these advances in the characterization of CPT2 and VLCAD deficiencies, few therapeutic approaches exist for these disorders. To date, the management of patients remains essentially supportive [START_REF] Angelini | Task force guidelines handbook: EFNS guidelines on diagnosis and management of fatty acid mitochondrial disorders[END_REF][START_REF] Spiekerkoetter | Current issues regarding treatment of mitochondrial fatty acid oxidation disorders[END_REF][START_REF] Vockley | Diagnosis and management of defects of mitochondrial beta-oxidation[END_REF]. A highcarbohydrate fat-restricted diet, associated with the prevention of fasting or exercise, has been the mainstay of treatment for many years. These preventive measures, which alleviate muscular symptoms in some myopathic patients, do not, however, prevent the onset of muscular alterations, which are associated with CPT2 or VLCAD deficiency.

As mentioned above, the literature data consistently suggest that some degree of correlation exists between the severity of clinical manifestations and the extent of the metabolic block as assessed in the patients' cells. Based on this, it can be surmised that treatments aimed at stimulating residual enzyme activity and FAO capacity might be of therapeutic value in CPT2 or VLCAD deficiency. In keeping with this hypothesis, we established that treatment by bezafibrate, a hypolipidemic drug known to activate the PPAR (Peroxisome Proliferator Activated Receptor) transcription factors, can correct the FAO defect in cells from patients with the mild form of these diseases [START_REF] Gobin-Limballe | Genetic basis for correction of verylong-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF][START_REF] Djouadi | Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders[END_REF][START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF]. Recently, a pilot trial of bezafibrate in myopathic CPT2-deficient patients suggested that this pharmacological approach could be successful in vivo [START_REF] Bonnefont | Bezafibrate for treatment of an inborn mitochondrial ß-oxidation defect[END_REF][START_REF] Bonnefont | Long-term follow-up of bezafibrate treatment in patients with the myopathic form of carnitine palmitoyltransferase 2 deficiency[END_REF].

These findings demonstrate that target-based therapy can be successful for the treatment of genetic FAO disorders, and prompted us to extend this paradigm to another key regulator of energy metabolism, namely the co-activator PGC-1α (Peroxisome Proliferator Activated Receptor Gamma Co-activator 1 alpha). PGC-1α acts as a co-activator of numerous transcription factors (PPARs, NRF 1 and 2 (nuclear respiratory factors), HNF4), and is a master regulator of mitochondrial biogenesis and mitochondrial oxidative metabolism [START_REF] Finck | PGC-1 coactivators: inducible regulators of energy metabolism in health and disease[END_REF][START_REF] Handschin | Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism[END_REF]. Accordingly, PGC-1α can be considered to be a highly relevant therapeutic target to stimulate residual metabolic capacities in FAO-deficient cells.

Recent studies have shown the importance of post-translational modifications in the control of PGC-1α activity, and suggest that post-translational activation of PGC-1α can be achieved by various classes of molecules, acting through different signaling pathways [START_REF] Handschin | Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism[END_REF]. In particular, PGC-1α can be de-acetylated by SIRT1, which results in PGC-1α activation [START_REF] Rodgers | Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[END_REF]. SIRT1 is a member of the sirtuin family of NAD-dependant histone deacetylases, and it is thought to be involved in the adaptation of cell metabolism to nutritional changes, which it senses via changes in the levels of NAD + (20).

Another activation mechanism could involve phosphorylation of PGC-1α, achieved by the AMPactivated protein kinase (AMPK). Activation by phosphorylation might provide another way to link PGC-1α activity to cell energy demand, since AMPK is considered to act as a fuel gauge activated by low energy status (20).

Resveratrol (RSV) is a natural polyphenolic compound, first isolated from various plant roots. RSV is, probably, one of the most studied natural compounds, because of its pleiotropic biological properties. Indeed, it is presently thought that RSV could delay the onset of some cancers, and have protective effects against common ageing disorders like type II diabetes, and cardiovascular, or neurodegenerative diseases, by its possible action on the cell cycle, apoptosis or inflammation, and by its antioxidant properties [START_REF] Baur | Therapeutic potential of resveratrol: the in vivo evidence[END_REF]. In addition, RSV has recently emerged as one of the most potent natural activators of SIRT1 (22-24), and various studies suggest that RSV might also act as an AMPK activator [START_REF] Dasgupta | Resveratrol stimulates AMP kinase activity in neurons[END_REF][START_REF] Hou | SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase[END_REF][START_REF] Zang | Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice[END_REF].

In this study, we investigated the potential of RSV to stimulate FAO capacities in a panel of patient's fibroblasts harboring CPT2 or VLCAD deficiencies, and we sought to delineate the molecular mechanisms underlying RSV metabolic effects in the patient cells.

RESULTS

Resveratrol can restore normal FAO flux in a panel of FAO-deficient fibroblasts.

In order to characterize the cellular response to RSV, palmitate oxidation rates were measured first in dose-response and time course experiments performed in control, VLCAD-, and CPT2-deficient fibroblasts. The optimal concentration of RSV was established by dose-response experiments employing 10 to 125µM of the compound in cells treated for 48h, as shown in Figure 1A. RSV induced a dose-dependant increase in palmitate oxidation rate in all the cell lines. In the patients' cells, the restoration of normal palmitate oxidation rates was observed at concentrations as low as 50µM RSV, and the maximal rates were reached at 75µM. At higher concentrations, RSV slowed down cell proliferation, as reflected by lower (-15% to -25%) protein levels. Based on these data, the effects of 75µM RSV were studied over a period of 12h to 72h. These time course experiments revealed significant increases in FAO after 24h of exposure, and maximal stimulation was reached at 48h (Figure 2B).

A panel of thirteen VLCAD-deficient cell lines with different genotypes (Table 1) was then treated for 48h with 75µM RSV (Figure 1C). Two patients (Patient 1 and 2) suffered from the severe form of the disease and, accordingly, exhibited very low residual palmitate oxidation in fibroblasts (around 5% of control values). In these severely deficient cell lines, RSV induced no significant change in FAO. In contrast, all the other patients (Patients 3-13) had been diagnosed with the milder, myopathic form of the disease and carried missense mutations that appeared compatible with the expression of low but significant residual FA oxidation (Figure 1C). Indeed, under basal conditions, the fibroblasts from patients 3 to 13 exhibited FAO rates ranging from 21% (Patients 3 and6 The effects of RSV were also tested in fibroblasts from the patients harboring different genotypes associated with the severe or with the mild form of CPT2 deficiency (Table 2). As shown in Figure 1D, fibroblasts from the severely affected Patient 14 exhibited a profound FAO deficiency, unchanged after treatment by RSV. In contrast, patients' fibroblasts with the mild form of CPT2-deficiency exhibited basal FAO rates ranging from 57% (Patient 15) to 72% (Patients 17 and19) of control values. Furthermore, treatment with RSV markedly stimulated FAO utilization, resulting in a complete correction of FAO rates in the five cell lines with mild CPT2 deficiency.

Resveratrol relieves the metabolic block by increasing the amount of mutated CPT2 or VLCAD protein.

Fibroblasts from control, VLCAD-or CPT2-deficient patients were then treated or not with RSV (75µM) for 48h and submitted to western-blot analysis. The amounts of VLCAD protein were significantly reduced in the fibroblasts, from -30% in Patient 10 up to -70% in Patient 4, compared to the normal level (Figure 2A). In control and VLCAD-deficient patients, RSV clearly up-regulated the levels of VLCAD proteins to amounts similar to, and eventually higher than, those observed in untreated control cells (2-to 3.5-fold, p<0.001). The same changes were observed in CPT2-deficient fibroblasts (Figure 2B), which exhibited reduced amounts of CPT2 protein under basal condition (-21% to -63%), and a strong increase (2.5-to 4.6-fold, p<0.001) in CPT2 protein levels in response to RSV treatment.

Quantitative RT-PCR was performed to determine the effects of RSV on mRNA levels of the VLCAD and CPT2 genes. As shown in Figure 2C and 2D, treatment with RSV resulted in a significant induction of VLCAD (+48% to +87%) and of CPT2 (+38% to +68%) gene expression in the control and the patients' cells.

Correction of FAO by Resveratrol requires SIRT1 and PGC-1α.

A number of biological effects of RSV have been proposed to involve SIRT1. This prompted us to investigate whether FAO induction by RSV in human fibroblasts was dependent on SIRT1. We first verified that control and FAO-deficient fibroblasts expressed detectable levels of SIRT1 protein under standard culture conditions (Figure 3B). Interestingly, it was found that exposure to RSV increased SIRT1 mRNA (from +30% to +40%, data not shown) and protein levels (from +33% to +67%) (Figure 3B) in control and in FAO-deficient fibroblasts. To address the possible role of SIRT1 in mediating the RSV effects, we studied the effects of Sirtinol, a known SIRT1 inhibitor. We used 40µM of the compound in the culture medium, a concentration determined in preliminary doseresponse experiments (data not shown). Control, VLCAD-and CPT2-deficient fibroblasts were treated with optimal doses of RSV, Sirtinol, or a combination of the two, for 48h and then FAO flux was measured (Figure 3A). Sirtinol alone had no significant effect on basal FAO rates. However, when tested in combination with RSV, Sirtinol appeared to fully suppress the FAO increases normally obtained in response to RSV. This inhibitory effect was observed in all the cell lines with the exception of Patient 12.

Because SIRT1 interacts with and activates PGC-1α, and because PGC-1α is a key co-activator of numerous genes involved in the FAO pathway, we investigated whether the effects of RSV on FAO were mediated by PGC-1α in human fibroblasts. To this end, we performed SiRNA experiments using control or PGC-1α SiRNA (Figure 4A). The Knockdown of PGC-1α totally prevented the FAO increase that normally occurs in response to RSV treatment, in control as well as FAO-deficient patients.

We next determined the effect of treatment with RSV on PGC-1α expression in control cells. PGC-1α mRNA was significantly induced (50% to 60%) by RSV treatment (data not shown), and was associated with an increase (1.7-fold, p<0.001) in PGC-1α protein (Figure 4B). We further determined, in parallel, whether PGC-1α could be activated at the posttranslational level by deacetylation, in response to RSV. This was shown to be the case since immunoprecipitation experiments revealed that exposure to RSV decreased the acetylation state of PGC-1α by 36%, consistent with its activation (Figure 4B).

Activation of AMPK does not impact FAO in control or patient cells.

Finally, since the effects of resveratrol could be mediated by AMPK, we asked the question as to whether the well-characterized AMPK activator, AICAR, was able to induce FAO in human fibroblasts. As can be seen Figure 5A, 500µM AICAR failed to increase the palmitate oxidation flux in control, VLCAD-or CPT2-deficient cells. Comparable results were obtained with 1mM AICAR, whereas higher concentrations of this compound tended to inhibit FAO (data not shown). To confirm these results, we tested in parallel the effects of A-769662, a specific and direct activator of AMPK [START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF]. A dose-response study was performed (Figure 5B) which showed no significant changes in palmitate oxidation up to 100µM. This dose was then tested in several other VLCAD-or CPT2deficient patients and it was found that A-769662 does not reproduce the effects of RSV on FAO in the various cells tested (Figure 5C). Altogether, these data show that none of the two AMPK activators tested is able to induce a stimulation of FAO in the control or in the FAO-deficient human fibroblasts.

DISCUSSION

RSV is a natural compound, produced by plants in response to bacterial or fungal attacks, which can be found in various food products such as red wine, grape juice or peanut butter. Besides its potential to inhibit carcinogenesis, or to protect against cardiac ischemic injuries (21), RSV also has been found to exert potent in vivo effects on energy and metabolic homeostasis. For example, it reduces fat deposits and improves insulin sensitivity in mice on a high-calorie diet [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. These metabolic effects target mitochondrial functions, since a global increase in mitochondrial oxidative metabolism and aerobic capacity has been reported in RSV-fed mice [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. However, the mechanisms have yet to be documented. In the present study we investigated the possible stimulatory effects of RSV on oxidative metabolism in human cells from FAO-deficient patients. To our knowledge, this is the first study that evaluates the potential of this compound in the field of inborn metabolic disorders.

Fibroblasts from patients with the myopathic form of VLCAD or CPT2 deficiency exhibited considerably lower VLCAD or CPT2 protein levels under basal conditions as compared to control fibroblasts. In the absence of RSV treatment, residual FAO capacities in the patients' cells varied from 20 to 70% of normal values. These features are typically associated with the presence of missense mutations which are characterized as mild, such as V174M (Patient 3), K264E (Patient 10) or V283A (Patient 5, 8, 13) in the VLCAD gene [START_REF] Gobin-Limballe | Genetic basis for correction of verylong-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF], or S113L (Patients 15-19) in the CPT2 gene [START_REF] Corti | Clinical features and new molecular findings in Carnitine Palmitoyltransferase II (CPT II) deficiency[END_REF][START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF].

Examination of these mutations in the crystal structures of the CPT2 or VLCAD proteins showed that none of them target active site residues [START_REF] Mcandrew | Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase[END_REF][START_REF] Rufer | The crystal structure of carnitine palmitoyltransferase 2 and implications for diabetes treatment[END_REF]. Consistent with this, previous studies have established that there are variable amounts of low residual VLCAD or CPT2 enzyme activities in cells from Patients 4, 5, 8, 9, 10, 13, or 15-17, respectively [START_REF] Gobin-Limballe | Genetic basis for correction of verylong-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF][START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF]. In contrast, cells from patients having the severe disease presentation harbor gene missense mutations which are in close vicinity to the enzyme active site (G185S, D328G), or which target highly conserved protein domains essential for catalysis (G441D), both of which produce an extremely unstable protein [START_REF] Gobin-Limballe | Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches[END_REF] having negligible residual FAO capacities. The results obtained in this study provide evidence for a marked stimulatory effect of RSV on patient cell FA utilization. Indeed, exposure to RSV restores normal FAO capacities in a panel of cells harboring mild CPT2 or VLCAD deficiency. In all cases, correction of the FAO flux, in response to RSV, could be ascribed to a marked induction of mutated CPT2 or VLCAD mRNA and protein levels. These findings are consistent with literature data reporting an up-regulation of MCAD (Medium Chain AcylCoA Dehydrogenase) gene expression level in the muscle of mice chronically fed RSV [START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. It should be noted that evidence for an RSV-induced stimulation of FAO have been, so far, based essentially on changes in the levels of ß-oxidation gene expression. In this regard, our study identifies new RSV targets in the mitochondrial ß-oxidation pathway, and it is the first to establish that exposure to RSV actually induces an increase in mitochondrial FAO flux in control human fibroblasts, and that exposure to RSV can restore normal FAO in genetically deficient cells from patients.

Our data suggest that these effects of RSV on mitochondrial ß-oxidation involve SIRT1. This appears clearly from the experiments using Sirtinol, a SIRT1 inhibitor, since this compound fully abrogated the FAO stimulation normally observed in fibroblasts exposed to RSV. These results are in agreement with data obtained in mouse embryonic fibroblasts (MEFs) from SIRT1 -/-mice, which showed that exposure to 50µM RSV increased MCAD mRNAs levels in SIRT1+/+ MEFs, but not in SIRT1-/-MEFs, thus indicating a pivotal role of SIRT1 in mediating RSV effects on mitochondrial ß-oxidation in mouse fibroblasts [START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. In vivo studies in mice indicate that SIRT1 activation by RSV results in a coordinated regulation of SIRT1 downstream targets [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. Among these targets, PGC-1α is considered to be a master regulator of mitochondrial energy metabolism, and, in particular, of fatty acid ß-oxidation [START_REF] Handschin | Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism[END_REF]. This led us to study the possible involvement of PGC-1α in the fibroblast response to RSV. In our experiments, disruption of PGC-1α expression by use of PGC-1α SiRNA abolished the stimulation of FAO by RSV. Furthermore, the level of acetylated PGC-1α was decreased in RSV-treated cells, consistent with a SIRT1-mediated activation of PGC-1α in RSV-treated cells.

RSV treatment also increased PGC-1α mRNA and protein levels. Altogether, these observations clearly suggest that there is a global increase in PGC-1α transcriptional activity, which could account for the up-regulation of the CPT2 and VLCAD genes in RSV-treated fibroblasts.

Interestingly, de-acetylation of PGC-1α, and increased levels of PGC-1α expression, also have been reported in the skeletal muscle of mice receiving chronic RSV supplementation [START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. Furthermore, it was shown in the same study that up-regulation of MCAD gene expression in C2C12 muscle cells exposed to RSV, was abolished by use of PGC-1α shRNA [START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. Importantly, recent studies clearly indicate that RSV can also mediate significant in vivo effects over short periods of time.

Thus, in high-fat fed mice, a 3-day treatment with SRT501 (a formulated RSV) led to an enhanced transcriptional activity of PGC-1α and the up-regulation of FAO genes in the liver [START_REF] Smith | Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo[END_REF]. A key role of the SIRT1-PGC-1α axis in regulating FAO also has been confirmed by other groups, in C2C12 cells [START_REF] Gerhart-Hines | Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha[END_REF] and in vivo, by the use of liver-specific SIRT1 knockout mice [START_REF] Purushotham | Hepatocytespecific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation[END_REF][START_REF] Rodgers | Fasting-dependent glucose and lipid metabolic response through hepatic sirtuin 1[END_REF]. Interestingly, we found that RSV treatment led to an increase in the levels of SIRT1 mRNA and protein in human cells. This is in keeping with the recent observation that the human SIRT1 gene is under control of PPARδ, a transcription factor known to be co-activated by PGC-1α [START_REF] Okazaki | PPARbeta/delta regulates the human SIRT1 gene transcription via Sp1[END_REF]. Altogether, our results show that RSVinduced stimulation of FAO in normal or FAO-deficient human fibroblasts require the presence of PGC-1α and SIRT1, in line with the results obtained in rodents.

In obese mice, the effect of RSV on energy metabolism has been proposed to be due partly to AMPK activation [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF]. RSV also has been reported to activate AMPK in neurons [START_REF] Dasgupta | Resveratrol stimulates AMP kinase activity in neurons[END_REF] and in HepG2 cells [START_REF] Hou | SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase[END_REF][START_REF] Zang | Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice[END_REF]. It is admitted at present that AMPK can activate PGC-1α by direct phosphorylation, thus leading to increase its transcriptional activity (39). It can thus be hypothesized that the effects of RSV observed in human fibroblasts might also involve AMPK. To address this issue, we evaluated the effects of two different AMPK activators: the well-known AICAR and the more specific AMPK activator A-769662 [START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF]. Neither of these AMPK activators induced changes in the FAO capacities of control or deficient cells, suggesting that AMPK cannot mediate the induction of FAO by RSV in human fibroblasts.

In conclusion, this study demonstrates that RSV can exert potent regulatory effects on a key mitochondrial energy producing pathway in human cells. It also provides the proof-of-concept that these effects could be relevant for the pharmacological correction of inherited FAO deficiencies. In common metabolic disorders (obesity, type II diabetes), RSV also could be beneficial because of its potential to correct mitochondrial dysfunctions associated with these disorders [START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF]. Other natural polyphenols, or so-called "nutraceuticals", structurally related to RSV (butein, piceatannol, quercetin,…) also have been shown to activate SIRT1 [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF] and could, therefore, elicit comparable metabolic effects, targeting mitochondrial oxidative metabolism. The involvement of SIRT1 in many biological processes, in particular ageing, has stimulated the quest for new SIRT1 activators and, recently, resulted in the identification of synthetic SIRT1 activators up to 1000-fold more potent than RSV [START_REF] Milne | Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes[END_REF]. Among these, SRT1720 was shown to increase oxidative metabolism by stimulating fatty acid utilization in rodent cells [START_REF] Feige | Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation[END_REF]. Other synthetic SIRT1 activators also are being tested in healthy volunteers or in patients with Type II diabetes (http://ClinicalTrials.gov). Finally, clinical trials are in progress to test the effects of RSV in patients with colon cancer or Alzheimer disease, and to assess the safety, pharmacokinetics and pharmacodynamics of SRT501, a formulation of RSV with improved bioavailability (http://ClinicalTrials.gov). Obviously, the intriguing biological properties of RSV raise challenging questions concerning the functioning of key regulatory mechanisms in cell energy metabolism, and they have stimulated the research for treatments for a variety of common diseases. In the same line of reasoning, the effects of RSV on mitochondrial function might, hopefully, stimulate the research in the expanding area of human genetic disorders of mitochondrial origin, for which therapeutic approaches remain quite limited.

MATERIALS AND METHODS

Patients and control Fibroblasts

The VLCAD-and CPT2-deficient human skin fibroblasts used for this study have been described previously [START_REF] Gobin-Limballe | Genetic basis for correction of verylong-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF][START_REF] Djouadi | Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders[END_REF][START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF], except Patient 19. Mutations, genotypes and phenotypes are given in Tables 1 and2.

Cell culture and treatments

Fibroblasts were grown in Ham'sF10 media (Invitrogen) with glutamine, 12% fetal bovine serum, 100U/ml penicillin, and 0,1mg/ml streptomycin under standard conditions, as previously described [START_REF] Djouadi | Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders[END_REF]. For treatment, the media were removed and the cells were incubated subsequently with fresh media containing the different effectors: 75µM Resveratrol (Cayman, Ann Arbor, MI, USA) and/or 40µM Sirtinol (Sigma, St Louis, MO, USA), or 500µM AICAR (5-Aminoimidazole-4carboxamide-1-ß-D-ribofuranoside; Sigma, St Louis, MO, USA), or 100µM A-769662 or with equivalent amounts of dimethyl sulfoxide (DMSO, vehicle). Cells were added for 48h.

Fatty acid oxidation measurement

Fatty acid oxidation was measured by quantitating the production of 3 H 2 O from (9,10-3 H) palmitate as described previously [START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF]. For each cell line, FAO measurements were performed in triplicate, and repeated in at least two independent experiments. The oxidation rates were expressed as nanomoles of 3 H fatty acid oxidized per hour par milligrams of cell protein (nmol 3 H FA/h/mg protein).

Western blot analysis

Western blots were performed as described elsewhere [START_REF] Djouadi | Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders[END_REF]. The following antibodies were used: anti-VLCAD (kindly provided by Dr S. Yamagushi, Japan), anti-SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ß-actin (Chemicon International, Temecula, USA).

Immunoreactive bands were scanned by densitometry with a computerized video densitometer and the results were expressed as arbitrary units normalized to the amount of ß-actin.

Real-Time quantitative PCR

Total RNA from fibroblasts was isolated using Trizol reagent (Invitrogen), treated with DNAse I (Ambion, Austin, TX, USA), and transcribed into cDNA as previously described [START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF]. The real-time quantification of the different transcripts was performed using the SYBR Green kit from Thermo Fisher Scientific (Epsom, UK) and a LightCycler (Roche Diagnostics) according to the manufacturer's instructions. The primers for CPT2, VLCAD, ß-actin and PGC-1α have been published previously [START_REF] Djouadi | Bezafibrate increases very-long-chain acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and is a potential therapy for fatty acid oxidation disorders[END_REF][START_REF] Bastin | Activation of peroxisome proliferator-activated receptor pathway stimulates the mitochondrial respiratory chain and can correct deficiencies in patients' cells lacking its components[END_REF]. The SIRT1 primers were: forward, GGGTACCGAGATAACCTTCTGT and reverse, TATCATCCAACTCAGGTGGAGG. The results are expressed in arbitrary units normalized to the amount of ß-actin.

SiRNA experiments

Small interfering RNA (siRNA) targeting PGC-1α sequences were synthesized by Sigma (Saint Louis, MO, USA), as well as control siRNAs. SiRNAs were transfected into fibroblasts using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

PGC-1α acetylation assays

PGC-1α lysine acetylation was analyzed by immunoprecipitation of PGC-1α from fibroblast nuclear extracts followed by western blot. Fibroblast nuclear extracts were obtained using a kit from Pierce Biotechnology (Thermo Fisher Scientific, Epsom, UK) according to the manufacturer's instructions. PGC-1α was immunoprecipitated using PGC-1α antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Total PGC-1α protein levels and acetylated forms were detected using specific antibodies for PGC-1α or acetyl lysine (Abcam, Cambridge, MA, USA).

LEGENDS TO FIGURES

  ) up to 72% (Patient 11) of control values. Exposure to 75µM RSV for 48h induced a strong and significant increase of FAO capacities in control fibroblasts, and in the eleven cell lines with mild VLCAD deficiency. The increases of palmitate oxidation in RSV-treated fibroblasts varied from +50% (Patient 11) up to +360% (Patient 12) as compared to vehicle-treated cells (p<0.001). Except for Patient 3, these inductions of FAO rates resulted in the restoration of normal palmitate oxidation in all the cell lines with mild VLCAD deficiency (Patients 4-13).

Figure 1 .

 1 Figure 1. Resveratrol increases palmitate oxidation in fibroblasts from control and FAO-

Figure 2 .

 2 Figure 2. Resveratrol increases VLCAD and CPT2 protein levels and gene expression.

Figure 3 .

 3 Figure 3. Resveratrol action is mediated through SIRT1.

Figure 4 .

 4 Figure 4. Resveratrol action involves PGC-1α.

Figure 5 .

 5 Figure 5. Resveratrol action is not mediated through AMPK.

Table 1

 1 Mutations of VLCAD-deficientPatients The numbering of nucleotides starts at the first adenine of the translation initiation codon. The numbering of amino acids starts at the first methionine encoded by the translation initiation codon. Phenotype at the time of diagnosis: S=severe; M=mild The numbering of nucleotides starts at the first adenine of the translation initiation codon. The numbering of amino acids starts at the first methionine encoded by the translation initiation codon. Phenotype at the time of diagnosis: S=severe; M=mild

	Patients

ACKNOWLEDGEMENTS

The authors thank Flore Aubey for technical assistance and Franck Brouillard for fruitful discussions. We are grateful to physicians from around the world who provided cell lines for this study. This work was supported by grants of the Association Française contre les Myopathies (AFM) and the Agence Nationale de la Recherche (ANR).

The authors declare no conflict of interest.

Expression of results and statistical analysis

Data are the means ± SD. Differences between groups were analyzed by one-way analysis of variance (ANOVA) and the Fisher test, or by the paired t test; p<0.05 was considered significant.


Fatima Djouadi

Florence Habarou

Carole Le Bachelier

Sacha Ferdinandusse

Dimitri J F Schlemmer

Jean-François Benoist

Audrey Boutron

Brage S Andresen

Gepke Visser

Pascale De Lonlay

Habarou Florence

Le Bachelier Carole

Ferdinandusse Sacha

Schlemmer Dimitri

François Jean

Benoist

Boutron Audrey

S Brage

Andresen

Visser Gepke

Pascale De Lonlay

Olpin Simon

Fukao Toshiyuki

Yamaguchi Seiji

W Arnold

Strauss

J A Wanders Ronald

Bastin F Jean
email: jean.bastin@inserm.fr

F Djouadi

C Habarou

J Le Bachelier

Bastin

Ronald J A Wanders

Robert Benoist Hôpital

Debré

Simon Olpin

Toshiyuki Fukao

Seiji Yamaguchi

Arnold W Strauss

Jean Bastin

Jean-Francois Benoist

Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mitochondrial trifunctional protein (MTP) is a large hetero-multimeric enzyme associated with the inner mitochondrial membrane that catalyzes the three final steps of long-chain mitochondrial fatty acid β-oxidation (FAO). MTP is composed of four α-and four β-subunits encoded by the HADHA and HADHB genes, respectively. Both genes are located head-tohead on chromosome 2 (2p23.3) and share a common promoter [START_REF] Orii | Genes for the human mitochondrial trifunctional protein alpha-and beta-subunits are divergently transcribed from a common promoter region[END_REF]. The alpha subunit carries the long-chain enoylCoA hydratase (LCEH) and the long-chain 3-hydroxyacylCoA dehydrogenase (LCHAD) activities, whereas the ß-subunit harbors the longchain 3-ketoacylCoA thiolase (LCKAT) activity [START_REF] Eaton | The mitochondrial trifunctional protein: centre of a beta-oxidation metabolon?[END_REF]. Disorders of the MTP complex, initially described in the 90's, are rare diseases with an estimated prevalence of 1:200000 newborns, classified in three groups [START_REF] Ijlst | Common missense mutation G1528C in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Characterization and expression of the mutant protein, mutation analysis on genomic DNA and chromosomal localization of the mitochondrial trifunctional protein alpha subunit gene[END_REF][START_REF] Fletcher | Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies[END_REF]. The majority of patients have an isolated LCHAD deficiency with moderate or no reduction in the hydratase and thiolase activities. Patients with decreased levels of all three enzymes activities (general MTP deficiency) are also observed [START_REF] Boutron | Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency[END_REF][START_REF] Sykut-Cegielska | Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening[END_REF]. Finally a small proportion of patients exhibit an isolated long-chain 3-ketothiolase deficiency [START_REF] Das | Isolated mitochondrial long-chain ketoacyl-CoA thiolase deficiency resulting from mutations in the HADHB gene[END_REF].

The molecular basis of these disorders is complex and only partially understood, and measurement of enzyme activities is the only means to distinguish between the various forms of the disorder [START_REF] Choi | Identification of novel mutations of the HADHA and HADHB genes in patients with mitochondrial trifunctional protein deficiency[END_REF][START_REF] Purevsuren | Study of deep intronic sequence exonization in a Japanese neonate with a mitochondrial trifunctional protein deficiency[END_REF][START_REF] Sperk | Outcome in six patients with mitochondrial trifunctional protein disorders identified by newborn screening[END_REF][START_REF] Fletcher | Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies[END_REF]. All patients with isolated LCHAD deficiency harbor at least one allele with the c. 1528G>C mutation, which affects the alpha subunit of the enzyme on position 474 (p.Glu474Gln), and lies within the LCHAD catalytic site [START_REF] Spiekerkoetter | General mitochondrial trifunctional protein (TFP) deficiency as a result of either alpha-or beta-subunit mutations exhibits similar phenotypes because mutations in either subunit alter TFP complex expression and subunit turnover[END_REF]. Other mutations in HADHA or HADHB gene often result in general MTP deficiency [START_REF] Spiekerkoetter | General mitochondrial trifunctional protein (TFP) deficiency as a result of either alpha-or beta-subunit mutations exhibits similar phenotypes because mutations in either subunit alter TFP complex expression and subunit turnover[END_REF]. To date, the Human Gene Mutation database reports 59 HADHA and 53 HADHB gene mutations, many of which have not been functionally characterized [START_REF] Boutron | Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency[END_REF][START_REF] Fletcher | Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies[END_REF].

These mutations are associated with a variety of clinical manifestations ranging from severe life-threatening phenotypes in the neonate to a mild adolescent-onset myopathic form with episodes of rhabdomyolysis [START_REF] Olpin | Pathophysiology of fatty acid oxidation disorders and resultant phenotypic variability[END_REF]). In addition to recapitulating symptoms of other FAO disorders, LCHAD deficiency is unique because patients often develop long-term peripheral neuropathy and retinopathy, with progressive loss of vision [START_REF] Fletcher | Observations regarding retinopathy in mitochondrial trifunctional protein deficiencies[END_REF][START_REF] Olpin | Pathophysiology of fatty acid oxidation disorders and resultant phenotypic variability[END_REF]. Altogether, deficiencies of the MTP complex are associated with a high mortality and morbidity and rank among the more severe FAO disorders [START_REF] Sykut-Cegielska | Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening[END_REF].

The common c. 1528G>C mutation, either in the homozygous or heterozygous state, is often associated with severe phenotypes and high mortality rates, but has also been reported in milder phenotypes [START_REF] Sykut-Cegielska | Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening[END_REF]. Overall, LCHAD and MTP deficiencies exhibit substantial molecular, biochemical, and clinical heterogeneity and no clear genotype-phenotype correlation [START_REF] Olpin | Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency[END_REF][START_REF] Boutron | Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency[END_REF][START_REF] Sykut-Cegielska | Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening[END_REF]. To date, management of the patients relies on nutritional supplementation with medium chain triglycerides, but no pharmacological treatments have been tested, either in pre-clinical or clinical studies [START_REF] Gillingham | Optimal dietary therapy of long-chain 3hydroxyacyl-CoA dehydrogenase deficiency[END_REF].

In recent years, we have shown that bezafibrate, a widely prescribed hypolipidemic drug, corrects, in some cases, CPT2 or VLCAD deficiency ex vivo in primary human cells [START_REF] Djouadi | PPARs as therapeutic targets for correction of inborn mitochondrial fatty acid oxidation disorders[END_REF]. In these experiments, the effects of bezafibrate were mediated by activation of Peroxisome Proliferator Activated Receptor (PPAR) nuclear receptors, which triggered a transcriptional stimulation of CPT2 and VLCAD gene expression. Data obtained in mice suggest that expression of HADHA and HADHB genes is controlled by the PPAR signaling pathway [START_REF] Aoyama | Altered constitutive expression of fatty acidmetabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha)[END_REF], and might therefore be stimulated in response to bezafibrate. There are, however, no data on a possible control of MTP by PPAR in human cells. In the present study, we therefore raised the question whether bezafibrate might be beneficial for improvement or correction of MTP deficiency in a panel of patient fibroblasts harboring different mutations of HADHA or HADHB gene.

Materials and methods

Cell culture

Human skin fibroblasts were obtained from cell repositories of different reference centers for the diagnosis of inborn metabolic diseases. All the patients' fibroblasts originated from individuals in which diagnosis of MTP deficiency had been previously established on the basis of clinical, biochemical and molecular data. This panel included all the cells available at the time of the study, without any selection, except for a few cells lines that were ruled out because of insufficient growth. The patient genotypes are given in Tables 1 and2. The present study included fibroblasts from 26 patients, homozygous or heterozygous for the common c.1528G>C HADHA gene mutation, or harboring various other HADHA or HADHB gene mutations. Control fibroblasts were obtained from three healthy individuals. Cells from patients or control individuals were grown in Ham's F10 media (Invitrogen, Cergy-Pontoise, France) with glutamine, 12% fetal bovine serum and 100 U/ml penicillin and 0.1 mg/ml streptomycin under standard conditions (5% CO 2 , 37°C). For treatment, cells were incubated in fresh media containing 400 µM bezafibrate (Sigma) or vehicle only (0.01% DMSO) for 48h.

Fatty acid oxidation (FAO) measurements FAO flux was determined by quantifying the production of 3 H 2 O from (9,10-3 H) palmitate (Perkin Elmer), in cultured fibroblasts, as previously described [START_REF] Manning | A comparison of[END_REF].

Palmitate bound to fatty acid-free albumin was used at the final concentration of 125 µM (60Ci/mmol). For each cell line, FAO measurements were performed in triplicate and repeated in two to three independent experiments. The oxidation rates were expressed as nmol of 3 H fatty acid oxidized per hour per mg of cell protein (nmol 3 H. FA/ h/ mg prot).

Acylcarnitine analysis

The method used has been described previously (Djouadi et al 2005). Briefly, fibroblasts were cultured in complete Ham's F10 medium containing 200µM palmitate and 400µM carnitine for 72 h at 37°C. The culture medium was collected, extracted, and analyzed for acylcarnitine content by electrospray MS-MS, using an API3000 triple quadrupole mass spectrometer (Sciex, Applied Biosystems, USA) detecting the precursors of an m/z ratio of 85, by reference to added internal deuterated standards. Long-chain acylcarnitines were quantified by reference to standard curves. The values were expressed relative to protein content determined by the Lowry method.

Enzyme activity

All enzyme activities were measured essentially as described previously using UHPLCanalysis to quantify substrate production and calculate enzyme activities [START_REF] Wanders | The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results[END_REF].

Western blot analysis

Mitochondria were isolated from fibroblasts using a mitochondrial/cytosol fractionation kit (Abcam, Cambridge, UK) according to the manufacturer's protocol. Cultured fibroblasts or isolated mitochondria from fibroblasts were harvested in RIPA buffer (50mM Tris, pH 7.5, 150mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 0.1% SDS, 10µg/ml leupeptin, 10µg/ml aprotinin, and 1mM phenylmethylsulfonyl fluoride) to prepare protein extracts. 25µg of total protein or 15 µg of mitochondrial protein per lane were resolved by 12% SDS-PAGE and transferred to Hybond-P PVDF membrane (Amersham Biosciences, Freiburg, Germany).

The following antibodies were used: mouse monoclonal anti-HADHA (dilution 1/1000) and anti-HADHB (dilution 1/500) (Abcam, Cambridge, UK) and mouse monoclonal anti-actin (dilution 1/2000) (Chemicon International, Temecula, USA). The primary antibodies were detected using horseradish peroxidase (HRP) conjugated anti-mouse IgG, and the chemiluminescent reagent, ECL (Amersham Biosciences, Freiburg, Germany).

Immunoreactive bands corresponding to the proteins were scanned by densitometry with a computerized video densitometer.

Statistical analysis

Data are the means ± SD. Unpaired t-tests were used for the comparisons between control and deficient fibroblasts, and paired t-test were used to analyze the effects of drug versus vehicle. P<0.05 was considered significant.

Results

The patient fibroblasts were first screened for their response to bezafibrate in tritiated palmitate oxidation assays. Basal oxidation rates in vehicle-treated fibroblasts revealed a marked long-chain fatty acid oxidation (FAO) deficiency in all the patients' fibroblasts harboring HADHA (Figure 1A) or HADHB (Figure 1B) mutations. Indeed, 16 of 26 patient cell lines (patients 2-4, 9-14, 17, 18, 20-24, 26) exhibited basal FAO rates that did not exceed 1 nmol 3 HFA/ h/ mg prot, indicating marked FAO deficiencies relative to the control value (4.8 nmol 3 HFA/ h/ mg prot). In 7 cell lines (patients 1, 7, 8, 15, 16, 19, 25), basal FAO rates were in the range of 1 to 2 nmol 3 HFA/ h/ mg prot, and only 2 patients' cell lines (patients 5 and 6) exhibited values close to 3 nmol 3 HFA/ h/ mg prot in the absence of treatment.

Exposure to 400µM bezafibrate for 48h resulted in a significant increase (+31%; p<0.001) in FAO capacity in control fibroblasts. There was substantial variability in the response to bezafibrate in our panel of patient cells harboring HADHA (Figure 1A) or HADHB (Figure 1B) gene mutations. Although many increases in FAO were found statistically significant, only those reaching at least 3 nanomol 3 HFA/ h/ mg prot, i.e 62% of the control value, were considered deserving of further investigation. Thus, in HADHA-deficient fibroblasts, only 5 of 18 cell lines (patients 1, 5, 6, 7, 8) fitted this criterion. Analysis of data also showed that none of the 4 cell lines homozygous for the common c.1528G>C HADHA gene mutation (patients 9-12) significantly responded to bezafibrate. Interestingly, however, 3 of the 6 bezafibrateresponsive fibroblasts (patient 6, 7, 8) harbored this c.1528G>C mutation in the heterozygous state. Marked increases in FAO capacity were observed in two HADHAdeficient cell lines (patient 7, 8) that initially exhibited very low basal FAO values (-75% compared to control), as well as in 3 cell lines (patients 1, 5, 6) with milder FAO deficiencies (-63 to -33% versus control). In this group of HADHA-deficient cells, the amplitude of FAO changes triggered by bezafibrate varied from 1.4-(patient 6) to 3.2-fold (patient 7). Overall, after treatment by bezafibrate, only one cell line (patient 5) was restored to control levels.

In HADHB-deficient fibroblasts (Figure 1B), all cell lines except patient 19 initially exhibited profound FAO deficiency (-80 to -95% relative to control) under standard culture conditions.

In this group, exposure to bezafibrate induced significant -but generally modest-increases in FAO flux values, which therefore remained quite deficient in most treated cells. By contrast, in patient 19, treatment with bezafibrate triggered a marked FAO stimulation, from 1.8 to 3.9 nanomol 3 HFA/ h/ mg prot (+117%, p<0.001). Thus, only Patient 19 was further investigated.

Next, we analyzed the effects of bezafibrate on LCHAD activity, an α-subunit function (Figure 2A), and LCKAT activity, the β-subunit enzyme (Figure 2B), in four fibroblasts in which FAO flux was significantly induced by the drug (patients 5, 6, 7, 19) and in 2 cells lines (patients 11 and 12, homozygous for c.1528G>C) that did not respond to bezafibrate. These cells were chosen because they reflect the two kinds of response to bezafibrate, and because enough cell material could be obtained to perform these assays. Short-chain hydroxyacyl-CoA dehydrogenase (SCHAD) activity was measured in parallel (Figure 2C). SCHAD was used as an internal standard to better evaluate the amplitude of defects in LCKAT and LCHAD activities in patient cells.

The results show (Figure 2A) that all the HADHA-deficient genotypes considered (patients 5, 6, 7, 11, and 12) were associated with a marked defect in LCHAD enzyme activity in the patient fibroblasts, which ranged from 14% (patient 5) down to 4% (patient 12), of control values. The residual levels of LCHAD enzyme activity were increased significantly, from +40% to +68% in three (patients 5, 6, and 7) of the five cell lines harboring HADHA mutations. Interestingly, bezafibrate induced no changes in LCHAD activity in both cell lines homozygous for c.1528G>C (patients 11 and 12). In HADHB-deficient patient 19 fibroblasts, treatment with bezafibrate resulted in a significant increase (+92%, p<0.001) in LCHAD residual enzyme activity. Finally, control fibroblasts exhibited a robust increase in LCHAD enzyme activity, from 82 to 122 nmol/min/mg prot (+46%, p<0.001), in response to bezafibrate.

Under basal conditions, LCKAT activity was unchanged (patient 12), or modestly reduced (patient 11) in the two cell lines homozygous for the c.1528G>C mutation, compared to control (Figure 2B). In patients 6 and 7 (c.1528G>C on one allele), LCKAT activity was close to 50% of normal value. In contrast, there was marked LCKAT deficiency in patient 5 (10% of control value) and in patient 19 (4% of control value) fibroblasts. As observed for LCHAD, LCKAT enzyme activity was markedly up-regulated in control fibroblasts (from 79 to 123 nmol/min/mg prot) after treatment with bezafibrate. Drug-induced increases in LCKAT were also observed in the two cell lines homozygous for the c.1528G>C mutation, which reached normal activity levels after treatment with bezafibrate, and in patient 6 and patient 7 fibroblasts, which reached 75% of normal values. In patient 5 and patient 19 fibroblasts, bezafibrate induced a small but significant, increase in LCKAT activity, but nevertheless both cell lines remained extremely deficient. Finally, SCHAD enzyme activity levels were normal, or close to normal, in all the patient cells, under basal conditions, indicating that mitochondrial short-chain fatty acid ß-oxidation is unaffected by MTP deficiency, and does not greatly vary among the various fibroblasts cell lines. In control and patient cells as well, SCHAD activity was generally induced (+34% to +64%) in response to bezafibrate (Figure 2C). Accumulation of long-chain hydroxyl-acylcarnitines is a hallmark of HADHA deficiency, which can be observed in vitro by incubating patient cells with long-chain fatty acids. We thus analyzed acylcarnitine production in fibroblasts with three distinct HADHA-deficient genotypes and investigated possible effects of bezafibrate (Figure 3). We chose two patients in which bezafibrate increased the FAO flux (patients 5 and 8) and one with severe LCHAD mutations, in which bezafibrate had no effect (patient 11). Under standard culture conditions, two hydroxylated acylcarnitine species, namely C14-OH and C16-OH, were present in large excess in patients' fibroblasts, compared to control, with the highest levels found in patient 11, homozygous for the c.1528G>C mutation. A modest decrease in these acylcarnitine species was observed in bezafibrate-treated patient 11 fibroblasts (-16% and -28%, respectively). Interestingly, however, exposure to bezafibrate resulted in a marked reduction of C14-OH and C16-OH acylcarnitines levels in patient 5 (-62% and -77%, respectively) and in patient 8 (-56% and -61%, respectively).

We then analyzed by western-blot the abundance of HADHA and HADHB protein levels in a panel of patient cells. A first set of experiments was performed using total protein extracts from control and HADHA-deficient fibroblasts. Unfortunately, and despite repeated tests, the commercially available antibody against the MTP alpha subunit did not allow detection of this protein in whole cell extracts. In contrast, the beta subunit protein could be immuno-detected and quantified in this material, and western blot analysis revealed a marked decrease in beta-subunit protein levels in HADHA-deficient fibroblasts, compared to control (figure 4A).

Interestingly, bezafibrate treatment led to increases in beta-subunit protein abundance in both control and HADHA-deficient fibroblasts. We were able to grow enough fibroblasts from patients 7 and 8 to isolate mitochondria and, in these preparations HADHA and HADHB could be detected in parallel (Figure 4B). This revealed extremely low residual alpha-subunit protein levels in both HADHA-deficient cells, and confirmed the relative diminution in betasubunit abundance in these cells, compared to control fibroblasts (Figure 4B). Quantification of the signals showed that bezafibrate significantly increased the abundance of alpha (x3) and beta (x1.5) subunit proteins in control fibroblasts, consistent with the up-regulation of the corresponding mRNA, by 1.3-and 1.6-fold for HADHA and HADHB genes, respectively (data not shown). In response to bezafibrate, parallel increases in the abundance of alpha and beta subunit proteins were also observed in mitochondrial fractions from both HADHAdeficient fibroblasts.

Discussion

The most common genetic defect affecting the mitochondrial trifunctional protein is the c1528G>C mutation in the HADHA gene, which results in LCHAD deficiency [START_REF] Spiekerkoetter | General mitochondrial trifunctional protein (TFP) deficiency as a result of either alpha-or beta-subunit mutations exhibits similar phenotypes because mutations in either subunit alter TFP complex expression and subunit turnover[END_REF][START_REF] Sykut-Cegielska | Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening[END_REF]. However, numerous other mutations affecting either the HADHA or the HADHB gene have now been described, leading to various forms of MTP deficiency [START_REF] Boutron | Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency[END_REF]. Many of these mutations have not been thoroughly characterized at the biochemical level, particularly as measurement of the three enzyme activities in patient cells is relatively complex, and since in vitro systems are difficult to use to analyze the effects of mutations, due to the hetero-multimeric state of the MTP. For these reasons, there is at the moment limited data on biochemical characterization of HADHA and HADHB deficiency in regards to the number of mutations and genotypes described. In order to address the functional consequences of various HADHA and HADHB mutations, we first performed parallel analysis of FAO capacities in 26 deficient cell lines, representing 16 different genotypes. In the absence of treatment with bezafibrate, the large majority of HADHA and HADHB fibroblasts exhibited extremely low (<20% of control or lower) residual FAO capacities, indicating that most mutations have severe negative consequences on the use of long-chain fatty acids as energy substrates. In particular, the four cell lines homozygous for the c1528G>C mutation, which completely abolishes LCHAD activity, were extremely deficient in this assay system. Accordingly, in our HADHA or HADHB-deficient fibroblasts, the defects in long-chain fatty acid ß-oxidation enzymes isoforms were not compensated by the activity of medium-or short-chain-specific isoforms, as suggested in some previous studies [START_REF] Olpin | Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency[END_REF]. We, and others, previously showed that the FAO assay provides a sensitive tool to evaluate the functional consequences of ß-oxidation enzyme deficiency on palmitate oxidation in patient fibroblasts. These marked ß-oxidation flux deficiencies in MTP-deficient fibroblasts are in line with the severe clinical manifestations of HADHA and HADHB defects, for which few treatments exist to date.

Previous studies in patient fibroblasts with CPT2 or VLCAD-deficiencies [START_REF] Djouadi | PPARs as therapeutic targets for correction of inborn mitochondrial fatty acid oxidation disorders[END_REF], established that FAO rate provided a sensitive parameter to reveal pharmacological stimulation of mitochondrial ß-oxidation by bezafibrate, or by other molecules [START_REF] Aires | Stilbenes and resveratrol metabolites improve mitochondrial fatty acid oxidation defects in human fibroblasts[END_REF]. In these studies, kinetics and dose-response experiments showed that maximal stimulation of FAO was reached using 400µM bezafibrate for 48 hours (Djouadi et al 2005).

In the present study, we therefore raised the question whether this treatment by bezafibrate could induce a stimulation of palmitate oxidation in our panel of HADHA or HADHB-deficient patient fibroblasts. The results obtained show that bezafibrate significantly improved the FAO deficiency in six cell lines. Specifically, after exposure to bezafibrate, fibroblasts from patients 1, 6, 7, 8, and 19 reached FAO values equivalent to 60-80% of normal value, and patient 5 was restored to control level. This reflected a stimulation of FAO capacities by bezafibrate varying from 1.3 to almost 3-fold in these cell lines. Interestingly, such pharmacological increases in FAO were observed in three cell lines (patient 6, 7, 8) heterozygous for the common c1528 G>C mutation. There were, to our knowledge, no data on possible effects of fibrates or PPAR agonists on the expression of mitochondrial trifunctional protein in human cells. Our western-blot studies show that, in control fibroblasts, both HADHA and HADHB protein levels were significantly up-regulated in response to bezafibrate. Additionally, both LCHAD and LCKAT enzyme activities were markedly induced by bezafibrate in control fibroblasts. Altogether, these data therefore indicate that mitochondrial trifunctional protein was up-regulated in bezafibratetreated control fibroblasts, which accounted for the parallel increases in LCHAD and LCKAT enzyme activities, and for the resulting up-regulation of FAO capacities.

We then addressed the question of whether pharmacological inductions of MTP protein subunits and enzyme activities could also be detected in those HADHA-deficient fibroblasts that were responsive to bezafibrate in palmitate oxidation assays. Quantification of HADHA protein was not possible in whole fibroblasts, but analysis of mitochondria-enriched fractions revealed extremely low levels of α-subunit protein and reduced levels of β-subunit proteins, in the HADHA-deficient fibroblasts. As observed in control fibroblasts, both the alpha and βsubunit proteins appeared up-regulated in response to bezafibrate. However, in line with the protein misfolding effects of mutations, the amounts of mutated alpha subunit in mitochondria of treated patient cells remained much lower than that found in mitochondria from treated control fibroblasts. It was difficult to precisely evaluate the residual HADHA protein levels in patients' cells and their changes in response to bezafibrate. Indeed, detection of mutant proteins by western-blot is often difficult in patient cells with FAO deficiency. This is why we preferentially used the tritiated palmitate oxidation rate as primary endpoint to assess the effects of bezafibrate in MTP-deficient fibroblasts.

In parallel, our results indicated significant, but modest, increases in LCHAD residual enzyme activity in those patient cell lines that responded to bezafibrate in FAO assays. Regarding LCKAT activity, the effects of bezafibrate varied among the different patient cells. In c.1528G>C homozygous genotypes, induction of LCKAT was in the same order of magnitude as in control fibroblasts factors, whereas, in contrast, a complete absence of response was observed in other deficient cells, which remained extremely deficient for this thiolase activity. As mentioned above, MTP has three distinct catalytic functions, and the changes in enzyme activities associated to various genotypes of MTP deficiency are still poorly delineated. In line with this, further studies would be needed to precisely evaluate the effects of bezafibrate on LCHAD and LCKAT in MTP-deficient fibroblasts with various genotypes. Finally, analysis of acylcarnitines in bezafibrate-responsive patient cells showed that accumulation of long-chain hydroxyl-acylcarnitines was markedly reduced after exposure to bezafibrate, but was not restored to control levels. Taken together, these data suggest that, in bezafibrate responsive fibroblasts, drug-induced increases in mutant proteins' levels and residual enzyme activities did occur, but were often not sufficient to fully restore the FAO flux, and hence to clear the accumulation of acylcarnitine species.

MTP is probably the more complex enzyme in the mitochondrial fatty acid ß-oxidation pathway, encoded by genes, assembled into a hetero-multimeric structure, and finally embedded in the inner mitochondrial membrane. Very little is known about the factors and signaling pathways that might be involved in regulating MTP expression. The work of Aoyama et al, in mice, established that expression of both HADHA and HADHB genes was strongly inducible in vivo in wild-type animals by treatment with WY14643, a potent PPAR alpha agonist, which led to up-regulation of both MTP subunits proteins. These effects were entirely PPAR-dependent since they were fully abolished in PPAR alpha knockout mice [START_REF] Aoyama | Altered constitutive expression of fatty acidmetabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha)[END_REF]. In our study, large scale sequencing of gene transcripts (RNAseq; data not shown) established that HADHA and HADHB gene expression levels were induced by bezafibrate (1.3-fold and 1.6-fold respectively), and we also found that the alpha-and ßsubunit protein levels, as well as the LCHAD and LCKAT activities were robustly induced, in response to bezafibrate, in human fibroblasts. It should be noted that RNAseq studies (Brage S. Andresen, personal communication) and previous data (Djouadi et al 2005), suggest that bezafibrate triggers a coordinate increase in gene expression of several other mitochondrial FAO enzymes. Likewise, this and previous studies indicate that, in control fibroblasts, pharmacological stimulation of long-chain FAO capacities by bezafibrate results from a coordinate up-regulation of multiple enzymes, including CPT2, involved in long-chain fatty acid import, as well as all the enzymes catalyzing the four steps of Lynen helix within the mitochondria, i.e. VLCAD and MTP.

In our study, however, treatment by bezafibrate often failed to improve FAO flux in HADHAor HADHB-deficient fibroblasts, in contrast with similar experiments performed in CPT2-or VLCAD-deficient fibroblasts. This could be explained by several factors. First, it should be recalled that, in the case of CPT2 or VLCAD deficiency, a response to bezafibrate was specifically observed in fibroblasts from patients with the mild muscular form of the disease, but not in cells from patients with the severe phenotype [START_REF] Djouadi | Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate[END_REF][START_REF] Gobin-Limballe | Genetic basis for correction of very-longchain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy[END_REF]. In both disorders, the positive pharmacological response was clearly ascribed to parallel increases in mutated gene mRNA, protein levels, and residual enzyme activity, resulting from the activation of PPAR nuclear receptors by bezafibrate [START_REF] Djouadi | PPARs as therapeutic targets for correction of inborn mitochondrial fatty acid oxidation disorders[END_REF]. Consistent with this scheme, no response was observed in patients harboring severe CPT2 or VLCAD gene mutations, which led to a complete defect of the corresponding enzyme, and therefore remained extremely deficient after exposure to bezafibrate.

Incidentally, this supports the notion that possible inductions by bezafibrate of other enzymes in the FAO pathway could not compensate for a complete deficiency at the CPT2 or VLCAD step. In the present study, we also found a number of cell lines that remained extremely deficient after treatment with bezafibrate. Accordingly, it can be thought that the HADHA and HADHB gene mutations in our panel of cells were generally severe (frameshift, non sense, splicing, etc…), and incompatible with the production of mutant protein, hence incompatible with pharmacological stimulation. Furthermore, while a clear-cut distinction between mild and severe phenotypes exists in CPT2 and VLCAD deficiency, there is a continuum of decreasing severity from the severe neonatal MTP phenotype to the mildest MTP phenotype, and possible correlations between phenotype, genotype and residual FAO capacities are not fully elucidated in this disorder.

Finally, some of the cell lines analyzed in the present study were functionally improved after treatment with bezafibrate. It appears unlikely that this effect was due to a compensatory stimulation of medium-or short-chain specific FAO enzymes, for several reasons. First, if this were the case, then stimulatory effects of bezafibrate would be found in all the HADHA-or HADHB-deficient treated cell lines, since medium-chain or short-chain specific FAO enzymes are normally operative in all the patients cells. Second, when bezafibrate treatment did induce FAO stimulation, it was also found to up-regulate the levels of mutant MTP alpha or MTP beta subunit, and it also tended to decrease the accumulation of long-chain hydroxyacylcarnitines, which are highly specific of MTP deficiency. Altogether, this clearly suggests that, when observed, the beneficial effects of bezafibrate on FAO capacities were primarily related to increases in residual MTP protein and enzyme activities.

Overall, the results obtained from control fibroblasts clearly identify both MTP alpha and beta subunits as possible targets of bezafibrate since, upon cell exposure to the drug, the protein abundance of each subunit, and the LCHAD and LKCAT activities, increased in parallel, and this was accompanied with a marked stimulation of FAO capacity. Nevertheless, the nature of several of the mutations in HADHA and HADHB appeared to result in little or no significant response to bezafibrate, most probably because these mutations abolished, or severely compromised, the production of mutant protein, as well as the expression of residual enzyme activity. As a result, positive effects of bezafibrate on FAO capacities were only observed in 23% of cases, in our panel of patient cells. Finally, the present study should be considered as exploratory, and has obvious limitations. In particular, considering the genetic heterogeneity of the disease, additional studies will be needed to fully analyze the potential of bezafibrate as a function of genotype, or to really evaluate the proportion of response to bezafibrate among all the different MTP-deficient genotypes. Furthermore, the possible relations between the disease phenotypes and the response to bezafibrate would also need to be investigated.

To conclude, it is worth mentioning that very recent pharmacological therapies developed for Duchenne myopathy [START_REF] Haas | European Medicines Agency review of ataluren for the treatment of ambulant patients aged 5 years and older with Duchenne muscular dystrophy resulting from a nonsense mutation in the dystrophin gene[END_REF] or cystic fibrosis [START_REF] Massie | Carrier screening for cystic fibrosis in the new era of medications that restore CFTR function[END_REF] are based on the restoration of low levels of the mutated protein function, and are effective only in a restricted number of disease genotypes [START_REF] Perez | Readthrough strategies for therapeutic suppression of nonsense mutations in inherited metabolic disease[END_REF]. Thus, although not necessarily applying to a majority of patients, the search of mutation-specific therapies can, in some cases, represent a valuable approach to progress in the treatment of rare genetic disorders. 1 and2

Figure legend Figure 1 .

 legend1 Figure legend

  . *p<0.05; ** p<0.01; *** p<0.001 compared with vehicle-treated fibroblasts.

Figure 2 .

 2 Figure 2. Measurements of MTP enzyme activities (A) Long-chain hydroxyacylCoA dehydrogenase, LCHAD, (B) Long-chain 3-ketoacylCoA thiolase, LCKAT, and (C) of shortchain hydroxyacylCoA dehydrogenase, SCHAD, in MTP-deficient patients fibroblasts, treated with bezafibrate (400 µM, 48h; dark bars), or with vehicle only (open bars). The results are mean ± SD. The genotypes are numbered as inTable 1. *p<0.05; ** p<0.01; *** p<0.001 compared with vehicle-treated fibroblasts.

Figure 3 .

 3 Figure 3. Acylcarnitine profiles in cultured control or patients' fibroblasts incubated with palmitate and carnitine for 72h, in the presence or absence of 400µM bezafibrate. The signals represent the molecular acylcarnitine ions detected by tandem mass spectrometry. Internal standards C8-IS: ( 2 H 3 )-octanoylcarnitine (m/z =292), C14-IS: ( 2 H 3 )-myristoylcarnitine (m/z =382); C16-IS: ( 2 H 3 )-palmitoylcarnitine (m/z=404). The histograms represent the quantification of C14-and C16-hydroxylated (OH)-acylcarnitines. Values are means ± SD. *** p<0.001 compared with vehicle-treated fibroblasts. m/z, mass/charge

Figure 4 .

 4 Figure 4. Quantification by western-blot analysis of (A) HADHB protein levels in whole protein extracts from control or patients fibroblasts, treated with bezafibrate (400 µM, 48h; dark bars), or with vehicle only (open bars) and normalized to actin abundance. (B) HADHA and HADHB protein levels in mitochondria-enriched extracts from control or patients fibroblasts, treated with bezafibrate (400 µM, 48h; dark bars), or with vehicle only (open bars) and normalized to porin abundance. ***p<0.001 compared with vehicle-treated fibroblasts.

,

Acknowledgements

This study was supported by grants of the Association Française contre les Myopathies (AFM).

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