ABSTRACT
Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.
Subject(s)
Caprylates/pharmacology , Fibroblasts/drug effects , Heptanoates/pharmacology , Lipid Metabolism, Inborn Errors/metabolism , Lipidomics/methods , Proteomics/methods , Acyl-CoA Dehydrogenase, Long-Chain/deficiency , Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Cardiolipins/metabolism , Cell Line , Female , Fibroblasts/metabolism , Genotype , Humans , Lipid Metabolism, Inborn Errors/genetics , Lipid Metabolism, Inborn Errors/pathology , Male , Mitochondria/drug effects , Mitochondria/metabolism , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Proteome/metabolism , Sphingolipids/metabolismABSTRACT
Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.
Subject(s)
Fatty Acids/metabolism , Fibroblasts/enzymology , Lipid Metabolism, Inborn Errors/enzymology , Lipidomics , Metabolome , Skin/enzymology , Acyl-CoA Dehydrogenase, Long-Chain/deficiency , Acyl-CoA Dehydrogenase, Long-Chain/genetics , Cardiolipins/metabolism , Carnitine O-Palmitoyltransferase/deficiency , Carnitine O-Palmitoyltransferase/genetics , Case-Control Studies , Cells, Cultured , Ceramides/metabolism , Female , Humans , Lipid Metabolism, Inborn Errors/genetics , Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase/deficiency , Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase/genetics , Male , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/genetics , Oxidation-Reduction , Sphingolipids/metabolism , Tandem Mass SpectrometryABSTRACT
In general, metabolic flexibility refers to an organism's capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial fatty metabolism: ß-oxidation and fatty acid biosynthesis. We focus specifically on two diseases: very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and malonyl-CoA synthetase deficiency (acyl-CoA synthetase family member 3 (ACSF3)) deficiency, which are both characterized by alterations in metabolic flexibility. On the one hand, in a mouse model of VLCAD-deficient (VLCAD-/-) mice, the white skeletal muscle undergoes metabolic and morphologic transdifferentiation towards glycolytic muscle fiber types via the up-regulation of mitochondrial fatty acid biosynthesis (mtFAS). On the other hand, in ACSF3-deficient patients, fibroblasts show impaired mitochondrial respiration, reduced lipoylation, and reduced glycolytic flux, which are compensated for by an increased ß-oxidation rate and the use of anaplerotic amino acids to address the energy needs. Here, we discuss a possible co-regulation by mtFAS and ß-oxidation in the maintenance of energy homeostasis.
Subject(s)
Congenital Bone Marrow Failure Syndromes/metabolism , Fatty Acids/metabolism , Lipid Metabolism, Inborn Errors/metabolism , Lipogenesis , Metabolic Diseases/metabolism , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Muscular Diseases/metabolism , Animals , Bacterial Proteins/metabolism , Coenzyme A Ligases/deficiency , Coenzyme A Ligases/metabolism , Congenital Bone Marrow Failure Syndromes/genetics , Congenital Bone Marrow Failure Syndromes/pathology , Fatty Acids/genetics , Humans , Lipid Metabolism, Inborn Errors/genetics , Lipid Metabolism, Inborn Errors/pathology , Metabolic Diseases/genetics , Metabolic Diseases/pathology , Mice , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Muscular Diseases/genetics , Muscular Diseases/pathologyABSTRACT
Very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) is the most common defect of long-chain fatty acid ß-oxidation. The recommended treatment includes the application of medium-chain triacylglycerols (MCTs). However, long-term treatment of VLCAD-/- mice resulted in the development of a sex-specific metabolic syndrome due to the selective activation of the ERK/mTORc1 signalling in females and ERK/peroxisome proliferator-activated receptor gamma pathway in males. In order to investigate a subsequent sex-specific effect of MCT on the lipid composition of the cellular membranes, we performed lipidomic analysis, SILAC-based quantitative proteomics and gene expression in fibroblasts from WT and VLCAD-/- mice of both sexes. Treatment with octanoate (C8) affected the composition of complex lipids resulting in a sex-specific signature of the molecular profile. The content of ceramides and sphingomyelins in particular differed significantly under control conditions and increased markedly in cells from mutant female mice but remained unchanged in cells from mutant males. Moreover, we observed a specific upregulation of biosynthesis of plasmalogens only in male mice, whereas in females C8 led to the accumulation of higher concentration of phosphatidylcholines and lysophosphatidylcholines. Our data on membrane lipids in VLCAD after supplementation with C8 provide evidence of a sex-specific lipid perturbation. We hypothesize a likely C8-induced pro-inflammatory response contributing to the development of a severe metabolic syndrome in female VLCAD-/- mice on long-term MCT supplementation.
Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain/genetics , Caprylates/pharmacology , Fibroblasts/drug effects , Gene Expression/drug effects , Lipidomics/methods , Proteomics/methods , Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Animals , Cells, Cultured , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Male , Mass Spectrometry , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , Sex FactorsABSTRACT
Malonyl-CoA synthetase (ACSF3) catalyzes the first step of the mitochondrial fatty acid biosynthesis (mtFASII). Mutations in ACSF3 cause CMAMMA a rare inborn error of metabolism. The clinical phenotype is very heterogeneous, with some patients presenting with neurologic manifestations. In some children, presenting symptoms such as coma, ketoacidosis and hypoglycemia are suggestive of an intermediary metabolic disorder. The overall pathophysiological mechanisms are not understood. In order to study the role of mtFASII in the regulation of energy metabolism we performed a comprehensive metabolic phenotyping with Seahorse technology proteomics in fibroblasts from healthy controls and ACSF3 patients. SILAC-based proteomics and lipidomic analysis were performed to investigate the effects of hypofunctional mtFASII on proteome and lipid homeostasis of complex lipids. Our data clearly confirmed an impaired mitochondrial flexibility characterized by reduced mitochondrial respiration and glycolytic flux due to a lower lipoylation degree. These findings were accompanied by the adaptational upregulation of ß-oxidation and by the reduction of anaplerotic amino acids as compensatory mechanism to address the required energy need. Finally, lipidomic analysis demonstrated that the content of the bioactive lipids sphingomyelins and cardiolipins was strongly increased. Our data clearly demonstrate the role of mtFASII in metabolic regulation. Moreover, we show that mtFASII acts as mediator in the lipid-mediated signaling processes in the regulation of energy homeostasis and metabolic flexibility.
Subject(s)
Coenzyme A Ligases/metabolism , Energy Metabolism , Fatty Acids/metabolism , Metabolism, Inborn Errors/metabolism , Mitochondrial Proteins/metabolism , Cells, Cultured , Coenzyme A Ligases/genetics , Fatty Acids/genetics , Glycolysis , Humans , Metabolism, Inborn Errors/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Oxidation-Reduction , Point MutationABSTRACT
Medium-chain-triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders (lcFAOD). Long-term treatment with MCT led to a sexually dimorphic response in the mouse model of very-long-chain-acyl-CoA-dehydrogenase-deficiency (VLCAD-/-) with the subsequent development of a metabolic syndrome in female mice. In order to evaluate the molecular mechanisms responsible for this sex specific response we performed a comprehensive metabolic phenotyping, SILAC-based quantitative proteomics and characterized the involved signaling pathways by western blot analysis and gene expression. WT and VLCAD-/- mice showed strong sex-dependent differences in basal metabolism and expression of proteins involved in the distinct metabolic pathways, even more prominent after treatment with octanoate. The investigation of molecular mechanisms responsible for the sexual dimorphisms delineated the selective activation of the ERK/mTORc1 signaling pathway leading to an increased biosynthesis and elongation of fatty acids in VLCAD-/- females. In contrast, octanoate induced the activation of ERK/PPARγ pathway and the subsequent upregulation of peroxisomal ßoxidation in males. We here provide first evidence that sex has to be considered as important variable in disease phenotype. These findings may have implications on treatment strategies in the different sexes.
