Metabolomic profile of H1N1-infected human lung epithelial A549 cell after mannose treatment via 13C MFA

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.

为进一步验证甘露糖是否可抑制葡萄糖供能的糖酵解过程，本研究采用1,2-13C₂-葡萄糖（1,2-13C2-glucose）与13C₆-甘露糖（13C6-mannose）标记细胞，开展13C标记代谢流分析（13C-labeled metabolic flux analysis, MFA），以此实现葡萄糖与甘露糖的代谢区分。结果显示，经甘露糖处理并感染H1N1病毒的A549细胞，其13C₂-葡萄糖-6-磷酸（13C2-glucose-6-phosphate, G6P）、13C₂-果糖-6-磷酸（13C2-fructose-6-phosphate, F6P）及13C₂-乳酸（13C2-lactic acid）的占比普遍降低。由此证实，甘露糖可减少葡萄糖来源的糖酵解通量。为探究甘露糖对三羧酸循环（tricarboxylic acid cycle, TCA）中间代谢物的影响，本研究沿用糖酵解分析的实验参数，同时开展代谢流分析与氧消耗速率检测。结果发现，甘露糖处理组的葡萄糖来源α-酮戊二酸（alpha-ketoglutarate, AKG）、琥珀酸（succinic acid）、富马酸（fumaric acid）及苹果酸（malic acid）的占比均有所升高，提示TCA循环与磷酸戊糖通路（pentose phosphate pathway, PPP）发生代谢重编程。

为系统探究甘露糖对糖酵解中间代谢物、磷酸戊糖通路（pentose phosphate pathway, PPP）及三羧酸循环（tricarboxylic acid cycle, TCA）的调控作用，本研究以人肺上皮A549细胞为模型，采用1,2-13C₂-D-葡萄糖（1,2-13C2-D-glucose）与13C₆-D-甘露糖（13C6-D-mannose）开展全范围代谢标记研究，通过同位素标记追踪各代谢通路中的碳原子流向。实验流程如下：将A549细胞置于含1,2-13C₂-D-葡萄糖与13C₆-D-甘露糖的培养基中培养，随后分别以单独葡萄糖或联合不同浓度甘露糖（0mM、5mM、25mM）预处理8小时；接着以感染复数（multiplicity of infection, MOI）为5的H1N1病毒感染细胞，感染24小时后收集样本。提取细胞内代谢物后，对各代谢物的质量同位素分布（mass isotopologue distribution, MID）进行分析，测定目标代谢物的同位素比例，并以对应代谢物的总池进行归一化处理。所有处理均重复5次（n=5）以保障实验结果的可靠性与重现性，最终结果以细胞数为基准进行归一化。统计分析采用单因素方差分析（one-way ANOVA）结合Dunnett事后检验，以H1N1-PBS组为对照进行多重比较，以此评估甘露糖处理对目标代谢通路的影响。本系列实验深入解析了H1N1感染背景下，葡萄糖与甘露糖对糖酵解、PPP及TCA循环的调控效应，为理解上述处理诱导的代谢重编程及其对病毒感染的潜在影响提供了关键实验依据。