Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Nuno Carinhas,Paula M Alves,Ana S Coroadinha,Daniel A M Pais,Ana P Teixeira,Alexey Koshkin,Paulo Fernandes,Manuel J T Carrondo

doi:10.1038/srep23529

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.

Highlights

Adenovirus vectors have attracted considerable interest as vaccines[1] and gene delivery systems for clinical applications[2], including cancer therapy[3]
We show that the metabolism in Madin-Darby Canine Kidney (MDCK) cells is characterized by a highly glycolytic metabolism and excretion of pyruvate-derived by-products in excess of glucose consumption, while the TCA cycle is significantly fueled by glutamine anaplerosis, entailing a particular wiring of fluxes at the level of pyruvate cycling
To gain a more detailed understanding of MDCK cells metabolism and the impact of Canine adenovirus vector type 2 (CAV2) production, we performed carbon labelling experiments combining two substrates, [1,2-13C]Glucose and [U-13C]Glutamine, which were administered in parallel cultures of both mock-infected and CAV2-infected cells (Fig. 1A)

Summary

Introduction

Adenovirus vectors have attracted considerable interest as vaccines[1] and gene delivery systems for clinical applications[2], including cancer therapy[3]. We show that the metabolism in MDCK cells is characterized by a highly glycolytic metabolism and excretion of pyruvate-derived by-products in excess of glucose consumption, while the TCA cycle is significantly fueled by glutamine anaplerosis, entailing a particular wiring of fluxes at the level of pyruvate cycling These cells displayed reductive AKG carboxylation activity under normal conditions. Upon CAV2 infection, our data reveals a significant upregulation of overall metabolic fluxes, including glycolysis, pentose-phosphate pathway (PPP), glutamine anaplerosis and, even more markedly, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, likely linked with increased lipogenesis. These results are discussed in the context of other viral infections and potential strategies for bioprocess optimization of CAV2 production are suggested

Methods

Results

Discussion

Conclusion