title: Effects of homocysteine on metabolic pathways in cultured astrocytes.
abstract: Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.
authors.affiliation: UCD School of Agriculture, Food Science and Veterinary Medicine, UCD Conway Institute, UCD Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Ying
authors.initials: Y
authors.lastname: Jin
authors.forename: Lorraine
authors.initials: L
authors.lastname: Brennan
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D006710:Homocysteine / Q000633:toxicity*; D009682:Magnetic Resonance Spectroscopy; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000187:drug effects / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000378:metabolism / Q000503:physiopathology; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013654:Taurine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D006710:Homocysteine; D013654:Taurine; D009249:NADP; C003949:hypotaurine; D017346:Protein Serine-Threonine Kinases; D011766:Pyruvate Carboxylase; D005947:Glucose; D002794:Choline