Inhibitory effect of 3-hydroxyacyl-CoAs and other long-chain fatty acidβ-oxidation intermediates on mitochondrial oxidative phosphorylation (original) (raw)
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Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity
Toxicological sciences : an official journal of the Society of Toxicology, 2018
Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be vali...
Evaluation of metabolic pathway activity in cultured skin fibroblasts and blood leukocytes
Clinica Chimica Acta, 1977
Screening for a variety of blocked catabolic mutants can be achieved in cultured skin fibroblasts and in peripheral blood leukocytes with a simple radioisotope method that is reliable and convenient. The method measures the incorporation into trichloroacetic acid (TCA) insoluble macromolecules of a "C-labelled intermediate in the pathway under question; incorporation of a 3H-labelled metabolite, not in the same pathway, is used as an internal control of metabolism in the cell population. The 14C/'H incorporation ratio is decreased in blocked pathways; use of the ratio method eliminates the delay required by radioautography (used in earlier adaptations of this method), the problems involved in CO2 collection, and the need to standardize cultures for cell number. This method has been used to identify cells with biochemical lesions in the oxidation of propionate, galactose, hypoxanthine and pyruvate; it has allowed 'us to identify a new variant of methylmalonicaciduria; we believe it can be extended to include other metabolites and pathways.
Molecular Genetics and Metabolism, 2004
The treatment for patients with genetic disorders of mitochondrial long-chain fatty acid b-oxidation is directed toward providing sufficient sources of energy for normal growth and development, and at the same time preventing the adverse effects that precipitate or result from metabolic decompensation. Standard of care treatment has focused on preventing the mobilization of lipids that result from fasting and providing medium-chain triglycerides (MCT) in the diet in order to bypass the long-chain metabolic block. MCTs that are currently available as commercial preparations are in the form of even-chain fatty acids that are predominately a mixture of octanoate and decanoate. Recently, the use of odd-chain fatty acids has been proposed as an alternative treatment. We have shown previously that the even-numbered medium-chain fatty acids (MCFAs) that are found in MCT preparations can reduce the accumulation of potentially toxic long-chain metabolites of fatty acid oxidation (FAO). In the current study, we undertook to determine if the same is true of odd-numbered MCFAs. We found that provision of odd-chain species does decrease the build-up of long-chain FAO intermediates in our in vitro skin fibroblast model, but to a lesser extent than even-numbered MCFAs.
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1986
In relation to the finding that human skin fibroblasts are capable of de novo ether phospholipid biosynthesis, we have studied the properties of acyl-CoA : dihydroxyacetone phosphate acyhransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Fu~~~ore, the transferase activity was found to be membrane-Lund and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA : dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.
Biochemical Journal, 1990
The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal β-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by β-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal β-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and...
Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability
2021
Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine as well as in the validation of ingredients for cosmetics. Given these cells’ short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in Normal Human Dermal Fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level (LGm), or its complete removal and replacement by galactose (OXPHOSm), always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm it was possible to observe the selective inhibition of mitochondrial ATP production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption...