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Mitochondria have emerged as key actors of innate and adaptive immunity. Mitophagy has a pivotal role in cell homeostasis, but its contribution to macrophage functions and host defense remains to be delineated. Here, we showed that lipopolysaccharide (LPS) in combination with IFN-[gamma] inhibited PINK1-dependent mitophagy in macrophages through a STAT1dependent activation of the inflammatory caspases 1 and 11. In addition, we demonstrated that the inhibition of mitophagy triggered classical macrophage activation in a mitochondrial ROS-dependent manner. In a murine model of polymicrobial infection (cecal ligature and puncture), adoptive transfer of Pink1-deficient bone marrow or pharmacological inhibition of mitophagy promoted macrophage activation, which favored bactericidal clearance and led to a better survival rate. Reciprocally, mitochondrial uncouplers that promote mitophagy reversed LPS/IFN-[gamma]-mediated activation of macrophages and led to immunoparalysis with impaired bacterial clearance and lowered survival. In critically ill patients, we showed that mitophagy was inhibited in blood monocytes of patients with sepsis as compared with nonseptic patients. Overall, this work demonstrates that the inhibition of mitophagy is a physiological mechanism that contributes to the activation of myeloid cells and improves the outcome of sepsis., Introduction Sepsis is one of the leading causes of morbidity and mortality in intensive care units (ICUs) (1). Strategies that blunt inflammatory responses have failed to improve survival, whereas those [...]

The CMS4 mesenchymal subtype of colorectal cancer (CRC) is associated with poor prognosis and resistance to treatment. The cellular prion protein PrPC is overexpressed in CMS4 tumors and controls the expression of a panel of CMS4-specific genes in CRC cell lines. Here, we sought to investigate PrPC downstream pathways that may underlie its role in CMS4 CRC. By combining gene set enrichment analyses and gain and loss of function approaches in CRC cell lines, we identify the integrin-linked kinase ILK as a proximal effector of PrPC that mediates its control on the CMS4 phenotype. We further leveraged three independent large CRC cohorts to assess correlations in gene expression pattern with patient outcomes and found that ILK is overexpressed in CMS4 mesenchymal tumors and confers a poor prognosis, especially when combined with high expression of the PrPC encoding gene PRNP. Of note, we discovered that the PrPC-ILK signaling axis controls the expression and activity of the tryptophan metabolizing enzyme indoleamine 2,3 dioxygenase IDO1, a key player in immune tolerance. In addition, we monitored alterations in the levels of tryptophan and its metabolites of the kynurenine pathway in the plasma of metastatic CRC patients (n = 325) and we highlight their prognostic value in combination with plasma PrPC levels. Thus, the PrPC-ILK-IDO1 axis plays a key role in the mesenchymal subtype of CRC. PrPC and IDO1-targeted strategies may represent new avenues for patient stratification and treatment in CRC. [ABSTRACT FROM AUTHOR]

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

Carnitine palmitoyltransferase-2 (CPT2) is a mitochondrial enzyme involved in long-chain fatty acid entry into mitochondria for their β-oxidation and energy production. Two phenotypes are associated with the extremely reduced CPT2 activity in genetically deficient patients: neonatal lethality or, in milder forms, myopathy. Resveratrol (RSV) is a phytophenol produced by grape plant in response to biotic or abiotic stresses that displays anti-oxidant properties, in particular through AP-1, NFκ B, STAT-3, and COX pathways. Some beneficiary effects of RSV are due to its modulation of microRNA (miRNA) expression. RSV can enhance residual CPT2 activities in human fibroblasts derived from CPT2-deficient patients and restores normal fatty acid oxidation rates likely through stimulation of mitochondrial biogenesis. Here, we report changes in miRNA expression linked to CPT2-deficiency, and we identify miRNAs whose expression changed following RSV treatment of control or CPT2-deficient fibroblasts isolated from patients. Our findings suggest that RSV consumption might exert beneficiary effects in patients with CPT2-deficiency. [ABSTRACT FROM AUTHOR]

Amyotrophic Lateral Sclerosis is an adult-onset neurodegenerative disease characterized by the specific loss of motor neurons, leading to muscle paralysis and death. Although the cellular mechanisms underlying amyotrophic lateral sclerosis (ALS)-induced toxicity for motor neurons remain poorly understood, growing evidence suggest a defective energetic metabolism in skeletal muscles participating in ALS-induced motor neuron death ultimately destabilizing neuromuscular junctions. In the present study, we report that a specific exercise paradigm, based on a high intensity and amplitude swimming exercise, significantly improves glucose metabolism in ALS mice. Using physiological tests and a biophysics approach based on nuclear magnetic resonance (NMR), we unexpectedly found that SOD1(G93A) ALS mice suffered from severe glucose intolerance, which was counteracted by high intensity swimming but not moderate intensity running exercise. Furthermore, swimming exercise restored the highly ALS-sensitive tibialis muscle through an autophagy-linked mechanism involving the expression of key glucose transporters and metabolic enzymes, including GLUT4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, GLUT4 and GAPDH expression defects were also found in muscles from ALS patients. Moreover, we report that swimming exercise induced a triglyceride accumulation in ALS tibialis, likely resulting from an increase in the expression levels of lipid transporters and biosynthesis enzymes, notably DGAT1 and related proteins. All these data provide the first molecular basis for the differential effects of specific exercise type and intensity in ALS, calling for the use of physical exercise as an appropriate intervention to alleviate symptoms in this debilitating disease. [ABSTRACT FROM AUTHOR]

Resveratrol is a natural polyphenolic compound produced by plants under various stress conditions. Resveratrol has been reported to exhibit antioxidant, anti-inflammatory, and anti-proliferative properties in mammalian cells and animal models, and might therefore exert pleiotropic beneficial effects in different pathophysiological states. More recently, resveratrol has also been shown to potentially target many mitochondrial metabolic pathways, including fatty acid β-oxidation or oxidative phosphorylation, leading to the up-regulation of the energy metabolism via signaling pathways involving PGC-1α, SIRT1, and/or AMP-kinase, which are not yet fully delineated. Some of resveratrol beneficial effects likely arise from its cellular effects in the skeletal muscle, which, surprisingly, has been given relatively little attention, compared to other target tissues. Here, we review the potential for resveratrol to ameliorate or correct mitochondrial metabolic deficiencies responsible for myopathies, due to inherited fatty acid β-oxidation or to respiratory chain defects, for which no treatment exists to date. We also review recent data supporting therapeutic effects of resveratrol in the Duchenne Muscular Dystrophy, a fatal genetic disease affecting the production of muscle dystrophin, associated to a variety of mitochondrial dysfunctions, which likely contribute to disease pathogenesis. [ABSTRACT FROM AUTHOR]

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 sulfoconjugates 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. [ABSTRACT FROM AUTHOR]

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by major fibro-fatty replacement of the right ventricle (RV). We hypothesized that changes in peroxisome proliferator-activated receptor (PPAR) signalling contributed to myocardium fatty accumulation and contractile dysfunction in ARVC. [ABSTRACT FROM PUBLISHER]

Carnitine palmitoyltransferase (CPT) deficiencies are common disorders of mitochondrial fatty acid oxidation. The CPT system is made up of two separate proteins located in the outer (CPT1) and inner (CPT2) mitochondrial membranes. While CPT2 is an ubiquitous protein, three tissue-specific CPT1 isoforms––the so-called “liver” (CPT1-A), “muscle” (CPT1B) and ‹brain› (CPT1-C) CPT1s––have been shown to exist. Amino acid and cDNA nucleotide sequences have been identified for all of these proteins. CPT1-A deficiency presents as recurrent attacks of fasting hypoketotic hypoglycemia. Twenty four CPT1A mutations have been reported to date. CPT1-B and -C deficiencies have not been hitherto identified. CPT2 deficiency has several clinical presentations. The “benign” adult form (more than 200 families reported) is characterized by episodes of rhabdomyolysis triggered by prolonged exercise. The prevalent S113L mutation is found in about 50% of mutant alleles. The infantile-type CPT2 presents as severe attacks of hypoketotic hypoglycemia, occasionally associated with cardiac damage commonly responsible for sudden death before 1 year of age. In addition to these symptoms, features of brain and kidney dysorganogenesis are frequently seen in the neonatal-onset CPT2 deficiency, almost always lethal during the first month of life. Around 40 CPT2 mutations (private missense or truncating mutations) have hitherto been detected. Treatment is based upon avoidance of fasting and/or exercise, a low fat diet enriched with medium chain triglycerides and carnitine. Prenatal diagnosis may be offered for pregnancies at a 1/4 risk of infantile/severe-type CPT2 deficiency. [Copyright &y& Elsevier]

The mitochondrial oxidation of fatty acids (FAO) is the main energy-producing pathway in skeletal and cardiac muscle. Starting from standard muscle biopsies (100–200 mg), we determined the optimal conditions of mitochondrial oxygen consumption by the FAO pathway, and in parallel we performed the isolation and primary culture of muscle cells to test their cellular FAO capacities. The determinations of maximal β-oxidation rates in the presence of palmitoyl-CoA or palmitoyl-l-carnitine (mean ± SEM: 32.5±2.0 and 34.1±1.3 nmol O2 min−1 mg−1 protein, n=16, respectively) provide a screening method of mitochondrial fatty acid transport system and intra-mitochondrial β-oxidation. We also determined the conditions of tritiated palmitate oxidation by human myoblasts (mean ± SEM: 6.6±0.1 nmol 3H fatty acid h−1 mg−1 protein, n=8), and show that β-oxidation defects can be detected in our experiments. Overall, we propose an original laboratory test to investigate FAO in human skeletal muscle and to screen for FAO disorders in myopathies and cardiomyopathies in human. [Copyright &y& Elsevier]

Examines the effects of fatty acids on mitochondrial Beta–oxidation enzyme gene expression in renal cell lines. Effect of fatty acids on gene expression of medium-chain acyl-CoA dehydrogenase; Occurrence of fatty acid-induced gene stimulation in the absence of cellular fatty acid accumulation; Development of fatty acid utilization in the kidney.

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

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A2 (TXA2). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid–produced aggregation. We also found that TXAS and TXA2 modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts. [ABSTRACT FROM AUTHOR]

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

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

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 + -dependent deacetylase and is critically dependent on NAD + 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. [ABSTRACT FROM AUTHOR]

Abstract: Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines. [Copyright &y& Elsevier]