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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; Mitochondrial respiratory chain (RC) disorders are the most prevalent inborn metabolic diseases and remain without effective treatment to date. Up-regulation of residual enzyme activity has been proposed as a possible therapeutic approach in this group of disorders. As resveratrol (RSV), a natural compound, was proposed to stimulate mitochondrial metabolism in rodents, we tested the effect of this compound on mitochondrial functions in control or in Complex I (CI)-or Complex IV (CIV)-deficient patients' fibroblasts. We show that RSV stimulates the expression of a panel of proteins representing structural subunits or assembly factors of the five RC complexes, in control fibroblasts. In moderate RC-deficient patients' cells, RSV treatment increases the amount of mutated proteins and stimulates residual enzyme activities. In these patients' cells, we establish that up-regulation of RC enzyme activities induced by RSV translates into increased cellular O 2 consumption rates and results in the correction of RC deficiencies. Importantly, RSV also prevents the accumulation of lactate that occurred in RC-deficient fibroblasts. Different complementary approaches demonstrate that RSV induces a mitochondrial biogenesis that might underlie the increase in mitochondrial capacities. Finally, we showed that, in human fibroblasts, RSV stimulated mitochondrial functions mainly in a SIRT1-and AMPK-independent manner and that its effects rather involved the estrogen receptor (ER) and estrogen-related receptor alpha (ERRa) signaling pathways. These results represent the first demonstration that RSV could have a beneficial effect on inborn CI and CIV deficiencies from nuclear origin, in human fibroblasts and might be clinically relevant for the treatment of some RC deficiencies.

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; 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.