(A) Mitochondrial stress tests quantifying oxygen consumption rate (OCR) by differentiating bone marrow stromal cells treated with vehicle or PTH for 2 hours with/without etomoxir (ETO), which was added 60 minutes before analysis. Oligomycin, FCCP, and rotenone/antimycin A were injected where shown by dashed lines. (B and C) OCR and extracellular acidification rate (ECAR) under basal conditions. (D–G) RNA sequencing was performed on differentiating osteoblast cultures treated with PTH for 24 hours. Volcano plot (D), gene enrichment for upregulated genes (E, numbers indicate the number of genes for each pathway), and heatmaps for genes associated with fatty acid (F) and glucose metabolism (G). For heatmaps, expression levels are shown using log₂-transformed counts per million normalized values. Asterisks represent genes with an adjusted P value less than 0.05. (H and I) Oxidation of oleate and glucose in osteoblast cultures. (J) Quantitative PCR (qPCR) analysis of fatty acid oxidation genes in the femurs of C57BL/6 mice 4 hours after treatment with vehicle or PTH (100 μg/kg BW, n = 5 mice/treatment). (K and L) Assessment of fatty acid uptake in the femurs assessed with bodipy-palmitate. Mice were injected with vehicle or PTH 1 hour prior to injection of bodipy-palmitate and labeling for 3 hours. (K) Micrographs represent composite images of a 3 × 2 matrix of 20× original magnification fields of view. Arrows denote positively labeled osteoblasts/bone-lining cells. Results are representative of 3 mice per treatment group. GP, growth plate. (L) Enlarged images of boxed regions from K. (M) qPCR analysis of Cpt2 mRNA. (N) Osteoblast differentiation was assessed by alkaline phosphatase and collagen staining after treatment with saline control or PTH for 7 days. All data are represented as mean ± SEM. Data were analyzed by ANOVA with Tukey’s multiple comparisons post hoc test (B) or unpaired Student’s t test (all other panels). * P < 0.05.