Translational Research Program of Pediatric Orthopedics, Department of Surgery, The Children's Hospital of Philadelphia; Department of Orthopedic Surgery, Tongji Hospital, Huazhong University of Science and Technology.
Translational Research Program of Pediatric Orthopedics, Department of Surgery, The Children's Hospital of Philadelphia.
J Vis Exp. 2022 Mar 23(181). doi: 10.3791/63568.
Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases. The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.
线粒体拥有三羧酸循环（TCA）和电子传递链（ETC）的机器，它们产生三磷酸腺苷（ATP）以维持能量平衡。葡萄糖、脂肪酸和氨基酸是大多数体细胞中驱动线粒体呼吸的主要能量底物。有证据表明，不同的细胞类型可能对某些底物有明显的偏好。然而，骨骼中各种细胞对不同底物的利用情况尚未得到详细研究。此外，由于细胞代谢适应生理和病理生理变化，直接评估骨骼细胞对底物的依赖性可能为骨病的发病机制提供重要见解。本方案基于氧化磷酸化过程中底物分子释放二氧化碳的原理。通过使用含有放射性标记碳原子（C）的底物，该方法为细胞培养中底物氧化速率提供了一种灵敏且易于使用的测定方法。以原代颅骨前成骨细胞与骨髓来源的巨噬细胞（BMM）为例的研究表明，两种细胞类型对主要底物的利用存在差异。
