Introduction: Cells use multilayered regulatory systems to respond adequately to changing environments or perturbations. Failure in regulation underlies cellular malfunctioning, loss of fitness, or disease. How molecular components dynamically interact to give rise to robust and adaptive responses is not well understood. Here, we studied how the model eukaryote Saccharomyces cerevisiae can cope with transition to high glucose levels, a failure of which results in metabolic malfunctioning and growth arrest. Methods: We combined experimental and modeling approaches to unravel the mechanisms used by yeast to cope with sudden glucose availability. We studied growth characteristics and metabolic state at population and single-cell levels (through flow cytometry and colony plating) of the wild type and of mutants unable to transit properly to excess glucose; such mutants are defective in trehalose synthesis, a disaccharide associated with stress resistance. Dynamic ¹³ C tracer enrichment was used to estimate dynamic intracellular fluxes immediately after glucose addition. Mathematical modeling was used to interpret and generalize results and to suggest subsequent experiments. Results: The failure to cope with glucose is caused by imbalanced reactions in glycolysis, the essential pathway in energy metabolism in most organisms. In the failure mode, the first steps of glycolysis carry more flux than the downstream steps, resulting in accumulating intermediates at constant low levels of adenosine triphosphate (ATP) and inorganic phosphate. We found that cells with such an unbalanced glycolysis coexist with vital cells with normal glycolytic function. Spontaneous, nongenetic metabolic variability among individual cells determines which state is reached and consequently which cells survive. In mutants of trehalose metabolism, only 0.01% of the cells started to grow on glucose; in the wild type, the success rate was still only 93% (i.e., 7% of wild-type yeast did not properly start up glycolysis). Mathematical models predicted that the dynamics of inorganic phosphate is a key determinant in successful transition to glucose, and that phosphate release through ATP hydrolysis reduces the probability of reaching an imbalanced state. ¹³ C-labeling experiments confirmed the hypothesis that trehalose metabolism constitutes a futile cycle that would provide proper phosphate balance: Upon a glucose pulse, almost 30% of the glucose is transiently shuttled into trehalose metabolism. Discussion: Our work reveals how cell fate can be determined by glycolytic dynamics combined with cell heterogeneity purely at the metabolic level. Specific regulatory mechanisms are required to initiate the glycolytic pathway; in yeast, trehalose cycling pushes glycolysis transiently into the right direction, after which cycling stops. The coexistence of two modes of glycolysis—an imbalanced state and the normal functional state—arises from the fundamental design of glycolysis. This makes the imbalanced state a generic risk for humans as well, extending our fundamental knowledge of this central pathway that is dysfunctional in diseases such as diabetes and cancer.

Background: Many details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations. Results: For most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points. Conclusions: Comparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: "Equation missing"<!-- image only, no MathML or LaTex -->. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h⁻¹) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.