The Gibbs free energy landscape based on liver metabolome revealed thermodynamic robustness against fasting and obesity.

Mammalian liver metabolism shifts substantially during fasting, yet the Gibbs free energy change of reaction (Δ r G'), a fundamental thermodynamic quantity that defines reaction driving force and constrains metabolic directionality, remains poorly characterized in intact organs. Here, we mapped the Δ r G' landscape of mouse liver glucose metabolism during fasting using absolute metabolite concentrations and GLEAM (Gibbs free energy of reaction landscape estimation from metabolome assisted by variance-covariance matrix), a computational method developed here. Despite metabolite fluctuations, Δ r G' landscapes remained robust, changing only modestly, even for reactions reversing direction between glycolysis and gluconeogenesis. Thermodynamically inferred potential rate-limiting steps were maintained during fasting. Enzyme-cost analysis further suggested a trade-off between thermodynamic robustness and enzyme requirements during glycolytic switching. Obese mouse liver also showed thermodynamic robustness despite metabolic disruption. Our framework provides a novel thermodynamic perspective on intact organ metabolism, revealing preservation of key thermodynamic characteristics across fasting and obesity.