Fig. 7.

Reactive oxygen species (ROS) production in primary osteocytes from GHRKO and control mice. Cells were seeded (0.4 × 10⁶ cells/mL) on collagen-coated black-walls 96-well plates and (A) incubated for 30 minutes with 2′,7′-dichlorofluorescin diacetate (DCFDA; 10 μg/mL). ROS was determined at excitation 495 nm and emission 525 nm (plate reader; Spectra Max5 M5, Molecular Devices with Softmax Pro software). Presented are ROS levels in the presence of 5 or 25 mM glucose in osteocytes from male and female mice, or (B) in the presence of insulin or insulin growth factor (IGF)-1 in osteocytes from male mice. (C) Cells were seeded as in (A) and incubated for 15 minutes with MitoSox (5 μM). Mitochondrial (mito) ROS levels in response to 0.1 μM rotenone were determined at excitation 510 nm and emission 580 nm. (D) Glutathione levels in osteocytes from young female and male mice in response to 0.1 μM rotenone were determined at excitation 394 nm and emission 490 nm. Data are presented as mean ± SEM of triplicates from three young (2-month-old) and three aged (2-year-old) control and GHRKO mice in each group. (E) Expression levels of oxidative stress defense enzymes (nuclear factor erythroid 2–related factor 2-Nrf2, superoxide dismutase-SOD, glutathione peroxidase-Gpx, catalase-CAT) in osteocytes from control and GHRKO mice. Significance (based on analysis of variance) as compared with young controls was accepted at p < 0.05. DCF = dichlorofluorescein; OD = optical density.