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 [1,2-13C]glucose and [U-13C]glutamine, we apply for the first time 13C-Metabolic flux analysis (13C-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 13C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. 13C-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.

13C‐metabolic flux analysis of human adenovirus infection: Implications for viral vector production

“…Still, AdV5 infection of EG cells caused an increase of this flux from 1.4 [0.8–2.1] to 3.0 [2.4–3.6] nmol/(10 6 cells × h). This increment matches a recent observation by our group of the upregulation of this flux after canine adenovirus vector infection, although in this case the cells had a much larger absolute flux (Carinhas et al, ). Moreover, it corroborates another recent observation of increased use of glutamine in reductive carboxylation after wild‐type human adenovirus infection (Thai et al, ).…”

“…The metabolic network used for flux estimation is similar to that recently published by our group to study MDCK cell metabolism under canine adenovirus vector infection (Carinhas et al, ). One difference is the omission of aspartate and asparagine transport rates in the present study given the absence of these amino acids in the culture medium or culture supernatants.…”

“…The other difference is the assumed cell dry weight of 271 pg (Carinhas et al, ), considerably lower than the 540 pg previously measured for MDCK cells. As previously discussed (Carinhas et al, , ), metabolic precursor requirements for adenoviral vector biomass production were omitted given their negligible impact on the material balance of producer cells. For convenience, all reactions with carbon atom transitions are specified in Appendix, together with lists of balanced and unbalanced metabolite pools.…”

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

“…A computational framework based on both metabolite and isotopomer balances is then used to integrate this data with experimentally determined uptake and secretion rates to estimate intracellular metabolic fluxes. This approach has been applied to study metabolic network operation and diagnosing of phenotypic perturbations in several biological systems [1,10,20,21], including primary astrocytic cultures [2].…”

Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication

“…These changes include the Warburg effect (aerobic glycolysis), amino acid catabolism (i.e., glutaminolysis [glutamate-driven anaplerosis]), lipid metabolism, activation of the pentose phosphate pathway, nucleotide biosynthesis, and amino acid biosynthesis (6–9). The effects are most noticeable at the genome replication stage (12 h post infection), and like the metabolic reprogramming that is seen in cancer cells and cells infected by some vertebrate viruses (10–13), the WSSV-induced metabolic changes benefit the virus by meeting the both its energy requirements and its biosynthetic needs.…”