13C stable isotope tracing reveals distinct fatty acid oxidation pathways in proliferative versus oxidative cells.

The TCA cycle serves as a central hub to balance catabolic and anabolic needs of the cell, where carbon moieties can either contribute to oxidative metabolism or support biosynthetic reactions. This differential TCA cycle engagement for glucose-derived carbon has been extensively studied in cultured cells, but the fate of fatty acid (FA)-derived carbons is poorly understood. To fill the knowledge gap, we have developed a strategy to culture cells with long-chain FAs without altering cell viability. By tracing ¹³C-FA, we show that FA oxidation (FAO) is robust in both proliferating and oxidative cells while the metabolic pathway after citrate formation is distinct. In proliferating cells, a significant portion of carbon derived from FAO exits canonical TCA cycle as citrate and converts to unlabeled malate in cytosol. Increasing FA supply or β-oxidation does not change the partition of FA-derived carbon between cytosol and mitochondria. Oxidation of glucose competes with FA-derived carbon for the canonical TCA pathway thus promoting FA carbon flowing into the alternative TCA pathway. Moreover, the coupling between FAO and the canonical TCA pathway changes with the state of oxidative energy metabolism.NEW & NOTEWORTHY By using ¹³C stable isotope-resolved metabolomics and FA-driven oxygen consumption rate analysis, our study provides novel insights into the fate of FA carbon through β-oxidation and downstream TCA cycle in proliferative and oxidative cells. Although both proliferative and oxidative cells demonstrate robust β-oxidation, they demonstrate distinct metabolic carbon fate downstream of citrate during TCA cycle oxidation. This differential TCA cycle engagement is likely to be important to balance catabolic and anabolic demands of the cell.