Effects of hindlimb unloading and bisphosphonates on the serum proteome of rats

Hindlimb unloading has been used as a model for bone loss associated with extended bed rest or space travel. However, this model also reduces muscle mass and influences other biological systems. To evaluate the impact of hindlimb unloading on bone and overall health, we applied 2-D gel electrophoresis (2-DE)-based proteomics to serum samples collected from 24 5-month-old female rats that were treated for 2 weeks under three conditions: control, hindlimb unloading (HU) and unloading plus bisphosphonate (HUA) (n = 8/group). Prior to the intervention, rats were injected with 3H-tetracycline to label bone surfaces. At the end of the experiment bone, urine, and serum samples were collected. As expected, HU reduced femur aBMD and BMC and increased daily urinary 3H-tetracycline (a measure of bone resorption rate) and these effects were reversed by bisphosphonate. In addition, serum osteocalcin and TRAP5b were decreased in the HUA compared to control and HU. Abundant proteins, albumin, IgG and transferrin were removed from serum samples prior to 2-DE analysis (n = 5 analytical replicates). Statistical analysis of spot intensities revealed 53 differentially expressed spots among the 3 groups. Cluster analysis shows that 30 spots reflect changes unique to the HU group (i.e. potential bone biomarkers), 6 unique to HUA (i.e. drug related), and 17 common to HU and HUA (e.g. potential mental stress or muscle loss markers). Spots were identified by LC-MS/MS after in-gel trypsin digestion and were found to relate to a variety of physiological functions.