Project Summary Metabolism is a dynamic network of biochemical reactions capable of adapting to changing environments. In changing glucose availability, glycolysis and gluconeogenesis support systemic glucose homeostasis. As glycolysis and gluconeogenesis operate several common reaction steps but in opposite directions, thermodynamics plays an important role in rapid oscillating between glycolysis and gluconeogenesis. In our parent grant, we made a fascinating observation that even though cell lines with constitutively activated Ras and Akt did not grow well under low-glucose high-lactate conditions, they proliferated rapidly under oscillatory conditions between abundant glucose and no glucose. We hypothesize that the deficiency in gluconeogenic enzyme expression caused by Ras and Akt renders these cells “inflexible” during the changing conditions and this inflexibility ironically contributes to their rapid utilization of glucose as soon as it becomes available. A corollary to this theory is that Ras- or Akt-activated cells may have more homogenous metabolism than healthy cells during oscillatory environments and the metabolic heterogeneity may undermine adaptive competitiveness in unstable fluctuating nutrient conditions. The goal of this administrative supplement application is to acquire BioSpa-Cytation (Agilent Technologies) to investigate the role of metabolic heterogeneity within cell populations in oscillatory nutrient environments. BioSpa-Cytation is an automated system that integrates incubator and multi- mode microscopy for studying 2D and 3D cultures in programmable dynamic environments. The overarching goal of the parent grant is to elucidate the regulatory mechanisms behind oscillation and coordination between glycolysis and gluconeogenesis by integrating metabolomics, fluxomics, and thermodynamics (mainly using mass spectrometry, isotope tracing, and quantitative modeling). The proposed equipment will provide the much- needed dynamic cell culturing and high-throughput microscopy capabilities, allow us to gain coherent microscopy and mass spectrometry insights, and ultimately lead to a greater success of the project in the parent grant.