Objective: To develop an accurate new method to measure the partitioning of adipocyte endogenous fatty acids among different metabolic pathways, a critical step toward understanding the regulatory mechanism by which fat disposition is modulated.

Research Methods and Procedures: Isolated primary rat adipocytes were pre‐incubated with isotope‐labeled fatty acids. This allows determination of the specific activity of labeled fatty acids in the endogenous lipid pool. After the removal of exogenous fatty acids, the disposition of endogenous fatty acids into the three major metabolic pathways, namely, oxidation, re‐esterification, and release into the medium, was measured independently. This was compared with the total lipolytic release of endogenous fatty acids, as measured by glycerol release. Adipocytes from normal fed and fasted animals were used to determine the effects of physiological variations on the metabolic fate of endogenous fatty acids.

Results: In normal fed animals, 0.2% of endogenous fatty acids were oxidized, 50.1% were released, and 49.7% were re‐esterified. Fasting doubled the partitioning of fatty acids toward oxidation (p < 0.05) in association with increased lipolysis (1.4‐fold increase) (p < 0.05). This effect was completely abolished by the addition of insulin to the cells (61% reduction) (p < 0.05).

Discussion: The endogenous fatty acids in adipocytes are actively oxidized. This process can be regulated by altered physiological conditions or by insulin. Over time, it is possible that a small shift of fatty acids toward oxidation could have a significant impact on body fuel economy. This hypothesis needs to be tested.