RESUMEN
OBJECTIVE: This study aims to elucidate the long-term benefit of newborn screening (NBS) for individuals with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiency, inherited metabolic diseases included in NBS programs worldwide. METHODS: German national multicenter study of individuals with confirmed LCHAD/MTP deficiency identified by NBS between 1999 and 2020 or selective metabolic screening. Analyses focused on NBS results, confirmatory diagnostics, and long-term clinical outcomes. RESULTS: Sixty-seven individuals with LCHAD/MTP deficiency were included in the study, thereof 54 identified by NBS. All screened individuals with LCHAD deficiency survived, but four with MTP deficiency (14.8%) died during the study period. Despite NBS and early treatment neonatal decompensations (28%), symptomatic disease course (94%), later metabolic decompensations (80%), cardiomyopathy (28%), myopathy (82%), hepatopathy (32%), retinopathy (17%), and/or neuropathy (22%) occurred. Hospitalization rates were high (up to a mean of 2.4 times/year). Disease courses in screened individuals with LCHAD and MTP deficiency were similar except for neuropathy, occurring earlier in individuals with MTP deficiency (median 3.9 vs. 11.4 years; p = 0.0447). Achievement of dietary goals decreased with age, from 75% in the first year of life to 12% at age 10, and consensus group recommendations on dietary management were often not achieved. INTERPRETATION: While NBS and early treatment result in improved (neonatal) survival, they cannot reliably prevent long-term morbidity in screened individuals with LCHAD/MTP deficiency, highlighting the urgent need of better therapeutic strategies and the development of disease course-altering treatment.
Asunto(s)
Cardiomiopatías , Errores Innatos del Metabolismo Lipídico , Miopatías Mitocondriales , Proteína Trifuncional Mitocondrial , Enfermedades del Sistema Nervioso , Rabdomiólisis , Humanos , Recién Nacido , Ácidos Grasos/metabolismo , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/terapia , Errores Innatos del Metabolismo Lipídico/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Proteína Trifuncional Mitocondrial/deficiencia , Lactante , Preescolar , NiñoRESUMEN
During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial ß-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or ß-oxidation activity.
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Proteínas Bacterianas , Proteína Trifuncional Mitocondrial , Mycobacterium tuberculosis , Humanos , Metabolismo de los Lípidos , Filogenia , Ubiquitinas , Proteína Trifuncional Mitocondrial/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Hypertriglyceridemia-induced acute pancreatitis (HTGP) is characterized by the acute and excessive release of FFA produced by pancreatic lipases. However, the underlying mechanisms of this disease remain poorly understood. In this study, we describe the involvement of the RNA binding protein hnRNPA2B1 in the development of HTGP. We used palmitic acid (PA) and AR42J cells to create a model of HTGP in vitro. RT-PCR and western blot analyses revealed a decrease in the level of hnRNPA2B1 protein but not mRNA expression in PA-treated cells. Further analyses revealed that hnRNPA2B1 expression was regulated at the post-translational level by neddylation. Restoration of hnRNPA2B1 expression using the neddylation inhibitor MLN4924 protected AR42J cells from PA-induced inflammatory injury by preventing NF-κB activation and restoring fatty acid oxidation and cell proliferation. Furthermore, RNA immunoprecipitation studies demonstrated that hnRNPA2B1 orchestrates fatty acid oxidation by regulating the expression of the mitochondrial trifunctional protein-α (MTPα). Administration of MLN4924 in vivo restored hnRNPA2B1 protein expression in the pancreas of hyperlipidemic mice and ameliorated HTGP-associated inflammation and pancreatic tissue injury. In conclusion, we show that hnRNPA2B1 has a central regulatory role in preventing HTGP-induced effects on cell metabolism and viability. Furthermore, our findings indicate that pharmacological inhibitors that target neddylation may provide therapeutic benefits to HTGP patients.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Hipertrigliceridemia , Pancreatitis , Enfermedad Aguda , Animales , Ciclopentanos , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ratones , Proteína Trifuncional Mitocondrial/metabolismo , FN-kappa B/metabolismo , Ácido Palmítico/farmacología , Pancreatitis/metabolismo , Pirimidinas , ARNRESUMEN
Long-chain 3-hydroxyacyl-CoA deficiency (LCHADD) and mitochondrial trifunctional protein (MTPD) belong to a group of inherited metabolic diseases affecting the degradation of long-chain chain fatty acids. During metabolic decompensation the incomplete degradation of fatty acids results in life-threatening episodes, coma and death. Despite fast identification at neonatal screening, LCHADD/MTPD present with progressive neurodegenerative symptoms originally attributed to the accumulation of toxic hydroxyl acylcarnitines and energy deficiency. Recently, it has been shown that LCHADD human fibroblasts display a disease-specific alteration of complex lipids. Accumulating fatty acids, due to defective ß-oxidation, contribute to a remodeling of several lipid classes including mitochondrial cardiolipins and sphingolipids. In the last years the face of LCHADD/MTPD has changed. The reported dysregulation of complex lipids other than the simple acylcarnitines represents a novel aspect of disease development. Indeed, aberrant lipid profiles have already been associated with other neurodegenerative diseases such as Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis and retinopathy. Today, the physiopathology that underlies the development of the progressive neuropathic symptoms in LCHADD/MTPD is not fully understood. Here, we hypothesize an alternative disease-causing mechanism that contemplates the interaction of several factors that acting in concert contribute to the heterogeneous clinical phenotype.
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Errores Innatos del Metabolismo Lipídico , Coenzima A , Ácidos Grasos/metabolismo , Humanos , Errores Innatos del Metabolismo Lipídico/genética , Proteína Trifuncional Mitocondrial/genética , Proteína Trifuncional Mitocondrial/metabolismo , Factores de Riesgo , EsfingolípidosRESUMEN
BACKGROUND: Mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies are rare fatty acid ß-oxidation disorders. Without dietary management the conditions are life-threatening. We conducted a systematic review to investigate whether pre-symptomatic dietary management following newborn screening provides better outcomes than treatment following symptomatic detection. METHODS: We searched Web of Science, Medline, Pre-Medline, Embase and the Cochrane Library up to 23rd April 2018. Two reviewers independently screened titles, abstracts and full texts for eligibility and quality appraised the studies. Data extraction was performed by one reviewer and checked by another. RESULTS: We included 13 articles out of 7483 unique records. The 13 articles reported on 11 patient groups, including 174 people with LCHAD deficiency, 18 people with MTP deficiency and 12 people with undifferentiated LCHAD/MTP deficiency. Study quality was moderate to weak in all studies. Included studies suggested fewer heart and liver problems in screen-detected patients, but inconsistent results for mortality. Follow up analyses compared long-term outcomes of (1) pre-symptomatically versus symptomatically treated patients, (2) screened versus unscreened patients, and (3) asymptomatic screen-detected, symptomatic screen-detected, and clinically diagnosed patients in each study. For follow up analyses 1 and 2, we found few statistically significant differences in the long-term outcomes. For follow up analysis 3 we found a significant difference for only one comparison, in the incidence of cardiomyopathy between the three groups. CONCLUSIONS: There is some evidence that dietary management following screen-detection might be associated with a lower incidence of some LCHAD and MTP deficiency-related complications. However, the evidence base is limited by small study sizes, quality issues and risk of confounding. An internationally collaborative research effort is needed to fully examine the risks and the benefits to pre-emptive dietary management with particular attention paid to disease severity and treatment group.
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Cardiomiopatías/diagnóstico , Errores Innatos del Metabolismo Lipídico/diagnóstico , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Miopatías Mitocondriales/diagnóstico , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades del Sistema Nervioso/diagnóstico , Rabdomiólisis/diagnóstico , Cardiomiopatías/etiología , Femenino , Humanos , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Masculino , Proteína Trifuncional Mitocondrial/efectos de los fármacos , Proteína Trifuncional Mitocondrial/metabolismoRESUMEN
BACKGROUND: Mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency are long-chain fatty acid oxidation disorders with particularly high morbidity and mortality. Outcome can be favorable if diagnosed in time, prompting the implementation in newborn screening programs. Sporadic cases missed by the initial screening sample have been reported. However, little is known on pitfalls during confirmatory testing resulting in fatal misconception of the diagnosis. RESULTS: We report a series of three patients with MTP and LCHAD deficiency, in whom diagnosis was missed by newborn screening, resulting in life-threatening metabolic decompensations within the first half year of life. Two of the patients showed elevated concentrations of primary markers C16-OH and C18:1-OH but were missed by confirmatory testing performed by the maternity clinic. A metabolic center was not consulted. Confirmatory testing consisted of analyses of acylcarnitines in blood and organic acids in urine, the finding of normal excretion of organic acids led to rejection and underestimation of the diagnosis, respectively. The third patient, a preterm infant, was not identified in the initial screening sample due to only moderate elevations of C16-OH and C18:1-OH and normal secondary markers and analyte ratios. CONCLUSION: Our observations highlight limitations of newborn screening for MTP/LCHAD deficiency. They confirm that analyses of acylcarnitines in blood and organic acids in urine alone are not suitable for confirmatory testing and molecular or functional analysis is crucial in diagnosing MTP/LCHAD deficiency. Mild elevations of primary biomarkers in premature infants need to trigger confirmatory testing. Our report underscores the essential role of specialized centers in confirming or ruling out diagnoses in suspicious screening results.
Asunto(s)
Biomarcadores/metabolismo , Cardiomiopatías/diagnóstico , Cardiomiopatías/metabolismo , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Miopatías Mitocondriales/diagnóstico , Miopatías Mitocondriales/metabolismo , Proteína Trifuncional Mitocondrial/deficiencia , Tamizaje Neonatal/métodos , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/metabolismo , Rabdomiólisis/diagnóstico , Rabdomiólisis/metabolismo , Humanos , Lactante , Recién Nacido , Proteína Trifuncional Mitocondrial/metabolismo , Oxidación-Reducción , Estudios RetrospectivosRESUMEN
The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid ß-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2ß2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPß decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.
Asunto(s)
Microscopía por Crioelectrón , Proteína Trifuncional Mitocondrial/ultraestructura , Humanos , Proteína Trifuncional Mitocondrial/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Mitochondrial trifunctional protein (MTP) plays a critical role in the oxidation of long-chain fatty acids. We previously reported that aging mice (>9 months old) heterozygous for an MTP defect (MTP+/-) develop nonalcoholic fatty liver disease (NAFLD). We tested whether a high-fat diet (HFD) accelerates NAFLD in young MTP+/-mice, and whether overexpression of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 3 (SIRT3) deacetylates MTP and improves mitochondrial function and NAFLD. Three-month-old WT and MTP+/- mice were fed HFD (60% cal fat) for 16 weeks and livers were assessed for fatty acid oxidation (FAO) and NAFLD. Compared with WT, MTP+/- mice displayed reduced hepatic SIRT3 levels and reduced FAO, with increased hepatic steatosis and the inflammatory marker CD68. Hepatic overexpression of SIRT3 in HFD-fed MTP+/- mice increased hepatic MTP protein levels at the posttranscriptional level. Immunoprecipitation of MTP from liver mitochondria followed by Western blot with acetyl-lysine antibody showed higher acetylation of MTP in MTP+/- compared with WT mice. Overexpression of SIRT3 in MTP+/- mice significantly reduced the acetylation of MTP compared with ß-galactosidase controls, increased mitochondrial FAO, and reduced hepatic steatosis, CD68, and serum ALT levels. Taken together, our data indicate that deacetylation of MTP by SIRT3 improves mitochondrial function and rescues NAFLD in MTP+/- mice.
Asunto(s)
Proteína Trifuncional Mitocondrial/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Acetilación , Animales , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Sirtuina 3/metabolismo , Triglicéridos/metabolismoRESUMEN
The antidepressant-like effect of fluoxetine (20 mg/kg i.p.) has been assessed using the forced swimming test (FST) in IRC (CD-1) mice exposed or not to a pretest session of different duration (5 or 20 min). The influence of the mitochondrial translocator protein (TSPO) activity on the antidepressant-like effect of fluoxetine (20 mg/kg i.p.) in the FST was also studied. The antidepressant-like effect of fluoxetine was observed only in mice subjected to a 5-min pretest session 24 h before the FST. The TSPO antagonist PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; 1 or 3 mg/kg i.p.] inhibited the antidepressant activity of fluoxetine in the FST. In the present study, fluoxetine or PK11195 was administered for a short duration. We suppose that the functional activity of TSPO may depend on a pretest session and that using this procedure is necessary to detect antidepressant activity of fluoxetine-like drugs.
Asunto(s)
Fluoxetina/metabolismo , Fluoxetina/farmacología , Proteína Trifuncional Mitocondrial/efectos de los fármacos , Animales , Antidepresivos/farmacología , Depresión/tratamiento farmacológico , Trastorno Depresivo/tratamiento farmacológico , Modelos Animales de Enfermedad , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Proteína Trifuncional Mitocondrial/metabolismo , Proteína Trifuncional Mitocondrial/fisiología , Actividad Motora/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Estrés Psicológico/tratamiento farmacológico , NataciónRESUMEN
BACKGROUND: Observational reports suggest that supplementation that increases citric acid cycle intermediates via anaplerosis may have therapeutic advantages over traditional medium-chain triglyceride (MCT) treatment of long-chain fatty acid oxidation disorders (LC-FAODs) but controlled trials have not been reported. The goal of our study was to compare the effects of triheptanoin (C7), an anaplerotic seven-carbon fatty acid triglyceride, to trioctanoin (C8), an eight-carbon fatty acid triglyceride, in patients with LC-FAODs. METHODS: A double blinded, randomized controlled trial of 32 subjects with LC-FAODs (carnitine palmitoyltransferase-2, very long-chain acylCoA dehydrogenase, trifunctional protein or long-chain 3-hydroxy acylCoA dehydrogenase deficiencies) who were randomly assigned a diet containing 20% of their total daily energy from either C7 or C8 for 4 months was conducted. Primary outcomes included changes in total energy expenditure (TEE), cardiac function by echocardiogram, exercise tolerance, and phosphocreatine recovery following acute exercise. Secondary outcomes included body composition, blood biomarkers, and adverse events, including incidence of rhabdomyolysis. RESULTS: Patients in the C7 group increased left ventricular (LV) ejection fraction by 7.4% (p = 0.046) while experiencing a 20% (p = 0.041) decrease in LV wall mass on their resting echocardiogram. They also required a lower heart rate for the same amount of work during a moderate-intensity exercise stress test when compared to patients taking C8. There was no difference in TEE, phosphocreatine recovery, body composition, incidence of rhabdomyolysis, or any secondary outcome measures between the groups. CONCLUSIONS: C7 improved LV ejection fraction and reduced LV mass at rest, as well as lowering heart rate during exercise among patients with LC-FAODs. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov NCT01379625.
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Caprilatos/uso terapéutico , Cardiomiopatías/tratamiento farmacológico , Ácidos Grasos/metabolismo , Errores Innatos del Metabolismo Lipídico/tratamiento farmacológico , Miopatías Mitocondriales/tratamiento farmacológico , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Rabdomiólisis/tratamiento farmacológico , Triglicéridos/uso terapéutico , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Adolescente , Adulto , Cardiomiopatías/metabolismo , Carnitina/metabolismo , Niño , Grasas de la Dieta/metabolismo , Método Doble Ciego , Ejercicio Físico/fisiología , Femenino , Humanos , Errores Innatos del Metabolismo Lipídico/metabolismo , Masculino , Persona de Mediana Edad , Miopatías Mitocondriales/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Oxidación-Reducción , Rabdomiólisis/metabolismo , Adulto JovenRESUMEN
Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a rare genetic disease. The LCHADD treatment is mainly based on special diet. In this diet, energy from long-chain triglycerides (LCT) cannot exceed 10%, however energy intake from the consumption of medium-chain triglycerides (MCTs) should increase. The daily intake of energy should be compatible with energy requirements and treatment should involve frequent meals including during the night to avoid periods of fasting. In fact, there are no recommendations for total content of LCT in all of the allowed food in the LCHADD diet. The aim of the study was to present a new method of diet composition in LCHADD with the use of blocks based on energy exchangers with calculated LCT content. In the study, the diet schema was shown for calculating the energy requirements and LCT content in the LCHADD diet. How to create the diet was also shown, based on a food pyramid developed for patients with LCHADD. The blocks will make it possible, in a quick and simple way, to create a balanced diet which provides adequate energy value, essential nutrients and LCT content. This method can be used by doctors and dietitians who specialize in treating rare metabolic diseases. It can also be used by patients and their families for accurate menu planning with limited LCT content.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/deficiencia , Cardiomiopatías/dietoterapia , Ingestión de Energía/fisiología , Errores Innatos del Metabolismo Lipídico/dietoterapia , Miopatías Mitocondriales/dietoterapia , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades del Sistema Nervioso/dietoterapia , Rabdomiólisis/dietoterapia , Triglicéridos/metabolismo , Adulto , Anciano , Cardiomiopatías/metabolismo , Niño , Preescolar , Dieta/métodos , Femenino , Humanos , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/metabolismo , Masculino , Persona de Mediana Edad , Miopatías Mitocondriales/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Rabdomiólisis/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Mitochondrial Trifunctional Protein deficiency (TFPD) is a severe genetic disease characterized by altered energy metabolism and accumulation of long-chain (LC) acylcarnitines in blood and tissues. This accumulation could impair the mitochondrial oxidative phosphorylation (OxPhos), contributing to the non-optimal outcome despite conventional diet therapy with medium-chain triglycerides (MCT). METHOD: Acylcarnitine and OxPhos parameters were measured in TFPD-fibroblasts obtained from 8 children and cultured in medium mimicking fasting (LCFA) or conventional treatment (MCT), with or without Etomoxir (ETX) an inhibitor of carnitine palmitoyltransferase 1 (CPT1) activity, and were compared to results obtained with fibroblasts from 5 healthy-control children. The effects of various acylcarnitines were also tested on control fibroblasts. RESULTS: In the LCFA-condition, TFPD-fibroblasts demonstrated a large accumulation of LC-acylcarnitines associated with decreased O2-consumption (63±3% of control, P<0.001) and ATP production (67±5%, P<0.001) without modification of coupling efficiency. A dose-dependent decrease in O2-consumption was reproduced in control fibroblasts by addition of increasing dose of LC-acylcarnitines, while it was almost preserved with MC-acylcarnitines. The MCT-condition reduced LC-acylcarnitine accumulation and partially improved O2-consumption (80±3%, P<0.01) in TFPD-fibroblasts. The addition of ETX in both LCFA- and MCT-conditions normalized acylcarnitine profiles and restored O2-consumption and ATP production at the same levels than control. CONCLUSION: Accumulation of LC-acylcarnitines plays a major role in the pathophysiology of TFPD, reducing OxPhos capacities. These deleterious effects could be partially prevented by MCT-therapy and totally corrected by ETX. Inhibition of CPT1 may be view as a new therapeutic target for patients with a severe form of TFPD.
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Cardiomiopatías/metabolismo , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Compuestos Epoxi/farmacología , Fibroblastos/metabolismo , Errores Innatos del Metabolismo Lipídico/metabolismo , Mitocondrias/metabolismo , Miopatías Mitocondriales/metabolismo , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades del Sistema Nervioso/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Rabdomiólisis/metabolismo , Cardiomiopatías/patología , Carnitina O-Palmitoiltransferasa/metabolismo , Femenino , Fibroblastos/patología , Humanos , Lactante , Recién Nacido , Errores Innatos del Metabolismo Lipídico/patología , Masculino , Mitocondrias/patología , Miopatías Mitocondriales/patología , Proteína Trifuncional Mitocondrial/efectos de los fármacos , Proteína Trifuncional Mitocondrial/metabolismo , Enfermedades del Sistema Nervioso/patología , Rabdomiólisis/patologíaRESUMEN
Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.
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Autofagia/fisiología , Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Animales , Alcaloides de Berberina/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subunidades de ProteínaRESUMEN
PURPOSE: To assess long-term effects of genotype on chorioretinopathy severity in patients with mitochondrial trifunctional protein (MTP) disorders. DESIGN: Retrospective case series. PARTICIPANTS: Consecutive patients with MTP disorders evaluated at a single center from 1994 through 2015, including 18 patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and 3 patients with trifunctional protein deficiency (TFPD). METHODS: Local records from all visits were reviewed. Every participant underwent a complete ophthalmic examination and was evaluated by a metabolic physician and dietitian. Nine patients underwent ancillary funduscopic imaging including optical coherence tomography (OCT) and OCT angiography. MAIN OUTCOME MEASURES: The primary outcome measure was best-corrected visual acuity at the final visit. Secondary outcome measures included spherical equivalent refraction, visual fields, electroretinography B-wave amplitudes, and qualitative imaging findings. RESULTS: Participants were followed up for a median of 5.6 years (range 0.3-20.2 years). The median age of LCHADD participants at initial and final visits was 2.3 and 11.9 years, whereas that for TFPD participants at initial and final visits was 4.7 and 15.5 years, respectively. Four long-term survivors older than 16 years were included (3 with LCHADD and 1 with TFPD). The LCHADD participants demonstrated a steady decline in visual acuity from an average of 0.23 logarithm of the minimum angle of resolution (logMAR; Snellen equivalent, 20/34) at baseline to 0.42 logMAR (Snellen equivalent, 20/53) at the final visit, whereas TFPD patients maintained excellent acuity throughout follow-up. Participants with LCHADD, but not TFPD, showed an increasing myopia with a mean decrease in spherical equivalent refraction of 0.24 diopters per year. Visual fields showed sensitivity losses centrally associated with defects on OCT. Multimodal imaging demonstrated progressive atrophy of the outer retina in LCHADD, often preceded by the formation of outer retinal tubulations and choriocapillaris dropout. Electroretinography findings support the more severe clinical profile of LCHADD patients compared with TFPD patients; the function of both rods and cones are attenuated diffusely in LCHADD patients, but are within normal limits for TFPD patients. CONCLUSIONS: Despite improved survival with early diagnosis, medical management, and dietary treatment, participants with the LCHADD subtype of MTP disorder continue to demonstrate visually disabling chorioretinopathy. Multimodal imaging is most consistent with choriocapillaris loss exceeding photoreceptor loss.
Asunto(s)
Cardiomiopatías/complicaciones , Enfermedades de la Coroides/etiología , Predicción , Errores Innatos del Metabolismo Lipídico/complicaciones , Miopatías Mitocondriales/complicaciones , Proteína Trifuncional Mitocondrial/deficiencia , Proteína Trifuncional Mitocondrial/genética , Enfermedades del Sistema Nervioso/complicaciones , Rabdomiólisis/complicaciones , Agudeza Visual , Adolescente , Adulto , Cardiomiopatías/diagnóstico , Cardiomiopatías/genética , Niño , Preescolar , Enfermedades de la Coroides/diagnóstico , Enfermedades de la Coroides/genética , Electrorretinografía , Femenino , Angiografía con Fluoresceína , Estudios de Seguimiento , Fondo de Ojo , Genotipo , Humanos , Lactante , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Miopatías Mitocondriales/diagnóstico , Miopatías Mitocondriales/genética , Proteína Trifuncional Mitocondrial/metabolismo , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/genética , Pronóstico , Estudios Retrospectivos , Rabdomiólisis/diagnóstico , Rabdomiólisis/genética , Tomografía de Coherencia Óptica , Campos Visuales , Adulto JovenRESUMEN
The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies.
Asunto(s)
Cardiomiopatías/metabolismo , Errores Innatos del Metabolismo Lipídico/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Miopatías Mitocondriales/metabolismo , Proteína Trifuncional Mitocondrial/deficiencia , Ácidos Mirísticos/farmacología , Enfermedades del Sistema Nervioso/metabolismo , Ácidos Palmíticos/farmacología , Rabdomiólisis/metabolismo , Animales , Calcio/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/fisiología , Proteína Trifuncional Mitocondrial/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , NADP/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ratas WistarRESUMEN
Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.
Asunto(s)
Señalización del Calcio , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Ácidos Mirísticos/metabolismo , Fosforilación Oxidativa , Ácidos Palmíticos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Cardiomiopatías/enzimología , Cardiomiopatías/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Errores Innatos del Metabolismo Lipídico/enzimología , Errores Innatos del Metabolismo Lipídico/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/enzimología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Miopatías Mitocondriales/enzimología , Miopatías Mitocondriales/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Proteína Trifuncional Mitocondrial/deficiencia , Proteína Trifuncional Mitocondrial/metabolismo , NADP/metabolismo , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/metabolismo , Especificidad de Órganos , Fosforilación Oxidativa/efectos de los fármacos , Ratas Wistar , Rabdomiólisis/enzimología , Rabdomiólisis/metabolismoRESUMEN
UNLABELLED: The course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid ß-oxidation. However, little is known about the effects of HCV on lipid ß-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and ß-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid ß-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid ß-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-of-function study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver. IMPORTANCE: HCV infection triggers metabolic alterations, which lead to significant disease outcomes, such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid ß-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid ß-oxidation, host cells were less responsive to the ability of exogenously added IFN-α to suppress HCV replication. This suggests that interference with lipid ß-oxidation may assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus-host interaction may lead to improvements in the current standard therapy.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hepacivirus/metabolismo , Hepatitis C/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Western Blotting , Línea Celular Tumoral , Vectores Genéticos/genética , Interacciones Huésped-Patógeno , Humanos , Interferón-alfa/metabolismo , Metabolismo de los Lípidos/fisiología , Luciferasas , Oxidación-Reducción , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the nigrostriatal pathway. The lipophile 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can cross the blood-brain barrier and is subsequently metabolized into toxic1-methyl-4-phenylpyridine (MPP(+) ), which causes mitochondrial dysfunction and the selective cell death of dopaminergic neurons. The present article reports the neuroprotective effects of silibinin in a murine MPTP model of PD. The flavonoid silibinin is the major active constituent of silymarin, an extract of milk thistle seeds, and is known to have hepatoprotective, anticancer, antioxidative, and neuroprotective effects. In the present study, silibinin effectively attenuated motor deficit and dopaminergic neuronal loss caused by MPTP. Furthermore, in vitro study confirmed that silibinin protects primary cultured neurons against MPP(+) -induced cell death and mitochondrial membrane disruption. The findings of the present study indicate that silibinin has neuroprotective effects in MPTP-induced models of PD rather than antioxidative or anti-inflammatory effects and that the neuroprotection afforded might be mediated by the stabilization of mitochondrial membrane potential. Furthermore, these findings suggest that silibinin protects mitochondria in MPTP-induced PD models and that it offers a starting point for the development of treatments that ameliorate the symptoms of PD.
Asunto(s)
Antioxidantes , Neuronas Dopaminérgicas/efectos de los fármacos , Intoxicación por MPTP/tratamiento farmacológico , Intoxicación por MPTP/patología , Mitocondrias/efectos de los fármacos , Actinas/metabolismo , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Proteínas de Unión al Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Embrión de Mamíferos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Mitocondrias/metabolismo , Proteína Trifuncional Mitocondrial/metabolismo , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Silibina , Silimarina/farmacología , Silimarina/uso terapéuticoRESUMEN
Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca(2+) retention capacity and ATP content, besides inducing swelling, cytochrome c release and H2O2 production in Ca(2+)-loaded mitochondrial preparations. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca(2+) uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca(2+), respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.