Metabolomic profile of H1N1-infected human lung epithelial A549 cell after mannose treatment via 13C MFA
Description
To further confirm if mannose inhibits glucose-fueled glycolysis, we conducted a 13C-labeled metabolic flux analysis (MFA) using 1,2-13C2-glucose and 13C6-mannose labeled cells, allowing for differentiation between glucose and mannose. We observed that the mannose-treated and H1N1-infected A549 cells exhibited generally lower proportions of 13C2-glucose-6-phosphate (G6P), 13C2-fructose-6-phosphate (F6P), and 13C2-lactic acid.Therefore, it is validated that mannose decreases glycolytic flow derived from glucose.To determine if mannose affects the TCA intermediates, both metabolic flux analysis and oxygen consumption rate measurement were conducted using the same experimental settings as for glycolysis analysis. We found that mannose-treated groups exhibited increased ratio of glucose-derived alpha-ketoglutarate (AKG), succinic acid, fumaric acid, and malic acid, indicating a reprograming of TCA cycle and pentose phosphate pathway. To investigate the impact of mannose on glycolysis intermediates, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, we conducted a comprehensive metabolic labeling study using 1,2-13C2-D-glucose and 13C6-D-mannose in human lung epithelial A549 cells. The labeling allowed us to track the carbon atoms in these metabolic pathways. A549 cells were cultured with 1,2-13C2-D-glucose and 13C6-D-mannose, and then pretreated with either glucose alone or in combination with mannose (0mM, 5mM, or 25mM) for 8 hours. Subsequently, the cells were infected with H1N1 virus at a multiplicity of infection (MOI) of 5 for 24 hours post-infection. Intracellular metabolites were extracted, and the mass isotopologue distribution (MID) of these metabolites was analyzed. The isotopologue ratio of the indicated metabolites was measured and normalized to the pool of the relevant metabolite. Each treatment was repeated five times (n=5) to ensure robustness and reliability of the findings. The obtained results were normalized based on cell number. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc test, with multiple comparisons against the H1N1-PBS group. This analysis allowed us to assess the impact of mannose treatment on the metabolic pathways of interest. These experiments provide valuable insights into the effects of glucose and mannose on glycolysis, the PPP, and the TCA cycle in the context of H1N1 infection. The results contribute to a deeper understanding of the metabolic reprogramming induced by these treatments and their potential implications for viral infections.