Stable Isotope Labeling to Track De Novo Fatty Acid Synthesis in Mouse Cartilage-Enriched Joint Tissue In Vivo: A Practical Workflow.

Cartilage is traditionally viewed as a metabolically quiescent tissue that relies primarily on exogenous lipid uptake to meet its energetic and structural demands. However, increasing evidence suggests that chondrocytes exhibit metabolically active behaviors, particularly under aging and pathological conditions. Here, we developed and applied an in vivo deuterium oxide (D2O)-based stable isotope labeling approach to directly quantify de novo fatty-acid synthesis in cartilage. Using gas chromatography-mass spectrometry (GC-MS), we tracked deuterium incorporation into palmitate in serum and articular cartilage from wild-type (WT) and cartilage-specific acetyl-CoA carboxylase 1 (ACC1) knockout mice. Progressive incorporation of deuterium into palmitate confirmed active fatty-acid synthesis in cartilage, with labeling kinetics distinct from those observed in serum, consistent with slower lipid turnover in the matrix-dense cartilage environment. Although fractional new synthesis of palmitate was comparable between genotypes during the labeling window, ACC1-deficient cartilage exhibited reduced palmitate abundance and decreased higher-order isotopomer enrichment at early labeling time points, indicating attenuated lipogenic flux. These findings provide direct in vivo evidence that chondrocytes possess intrinsic lipogenic capacity and that ACC1 contributes to the maintenance of the cartilage lipid pool. Together, this study establishes D2O-based isotope tracing as a robust and quantitative framework for assessing cartilage lipid metabolism in vivo and reveals de novo lipogenesis as an active metabolic process in cartilage. CLINICAL SIGNIFICANCE: This approach offers a valuable platform for investigating lipid dysregulation in osteoarthritis and other joint diseases.