Entropy Production Rates in Living Cells Under Metabolic Stress

Povzetek

Living cells maintain their highly ordered internal state only by continuously producing entropy in their surroundings,
yet how this entropy-production rate responds to graded metabolic stress, and whether it does so proportionally or
reveals distinct regimes, has been characterized in comparatively few quantitative studies. We measured entropy
production rate in cultured HeLa and primary fibroblast cell populations (n = 36 independent culture replicates per
cell type) using isothermal microcalorimetry combined with respirometry-derived oxygen-consumption and
lactate-production measurements, across five levels of metabolic stress induced by graded mitochondrial uncoupler
exposure (0, 0.25, 0.5, 1.0, and 2.0 micromolar FCCP). Baseline entropy production rate under unstressed
conditions averaged 1.84 +/- 0.21 pW/K per cell (mean +/- SD) in HeLa cells and 1.31 +/- 0.18 pW/K per cell in
fibroblasts, a significant difference between cell types (p < 0.001, two-sample t-test). Entropy production rate rose
monotonically with uncoupler concentration in both cell types, reaching 3.62 +/- 0.34 pW/K per cell in HeLa cells at
the highest dose tested, a 97% increase over baseline, and plateauing between the 1.0 and 2.0 micromolar doses
(3.58 versus 3.62 pW/K, difference not significant, p = 0.41), indicating a saturating rather than linear
stress-response relationship. The fraction of total entropy production attributable to oxidative (respiratory) versus
glycolytic (lactate-producing) pathways shifted substantially with stress level, from 76% oxidative at baseline to 54%
oxidative at the highest uncoupler dose, as cells compensated for impaired oxidative phosphorylation efficiency by
upregulating glycolytic flux. Entropy production rate per unit of ATP synthesized increased from 0.29 pW/K per
ATP/s at baseline to 0.61 pW/K per ATP/s at the highest dose, a 110% rise, demonstrating that cells under
uncoupling stress produced substantially more entropy per unit of useful chemical work performed, consistent with
reduced thermodynamic efficiency of energy transduction under mitochondrial stress. A linear mixed-effects model
confirmed a significant dose-by-cell-type interaction (p = 0.008), with HeLa cells showing a proportionally larger
relative increase in entropy production than fibroblasts across the dose range tested. These results provide a
quantitative, mechanistically decomposed picture of how cellular entropy production scales with graded metabolic
stress and suggest that entropy-production rate and its oxidative-glycolytic decomposition could serve as sensitive,
thermodynamically grounded biomarkers of mitochondrial dysfunction.