Comprehensive quantification of metabolic flux during acute cold stress in mice

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough un-derstanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quanti-tative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and mini-mally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling ana-lyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key func-tional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.