Glucose Challenge Uncovers Temporal Fungibility of Metabolic Homeostasis over a day:night cycle.

Rhythmicity is a cornerstone of behavioral and biological processes, especially metabolism, yet the mechanisms behind metabolite cycling remain elusive. This study uncovers a robust oscillation in key metabolite pathways downstream of glucose in humans. A purpose-built C-glucose isotope tracing platform was used to sample every 4h and probe these pathways, revealing a striking peak in biosynthesis shortly after lights-on in wild-type flies. A hyperactive mutant () demonstrates increased Krebs cycle labelling and dawn-specific glycolysis labelling. Surprisingly, neither underlying feeding rhythms nor the presence of food availability explain the rhythmicity of glucose processing across genotypes, suggesting a robust internal mechanism for metabolic control of glucose processing. These results align with clinical data highlighting detrimental effects of mistimed energy intake. Our approach offers a unique insight into the dynamic range of daily metabolic processing and provides a mechanistic foundation for exploring circadian metabolic homeostasis in disease contexts.