title: Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.
abstract: Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Nuno
authors.initials: N
authors.lastname: Carinhas
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Daniel A M
authors.initials: DA
authors.lastname: Pais
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Alexey
authors.initials: A
authors.lastname: Koshkin
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Fernandes
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Ana S
authors.initials: AS
authors.lastname: Coroadinha
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Teixeira
mesh_terms: D006516:Adenoviruses, Canine / Q000502:physiology*; D000818:Animals; D002247:Carbon Isotopes / Q000493:pharmacokinetics; D049109:Cell Proliferation; D002472:Cell Transformation, Viral; D004285:Dogs; D005786:Gene Expression Regulation; D005947:Glucose / Q000493:pharmacokinetics*; D005973:Glutamine / Q000493:pharmacokinetics*; D006019:Glycolysis; D050155:Lipogenesis; D061985:Madin Darby Canine Kidney Cells / Q000378:metabolism / Q000821:virology*; D064688:Metabolic Flux Analysis / Q000379:methods*; D010427:Pentose Phosphate Pathway
chemical_list: D002247:Carbon Isotopes; D005973:Glutamine; D005947:Glucose