Testosterone

Blood was acquired prior to surgery and biweekly thereafter, as described above. Serum hormones were measured in duplicate on a single plate and are reported as change from baseline, except for Testosterone which is reported as actual concentration because insufficient baseline sera was available to calculate percentage change. Procollagen type 1 N-terminal (P1NP) and osteocalcin (bone formation markers) and tartrate-resistant acid phosphatase 5b (TRAP5b, an osteoclast derived bone resorption marker) were determined by EIA with intra-assay CVs < 7.4% (IDS, Fountain Hills, AZ). Testosterone was evaluated by EIA with intra-assay CV < 9% (ALPCO, Salem, NH). Leptin was determined by ELISA with intra-assay CV < 2.5% (Millipore, MA). Sclerostin was assessed by ELISA with intra-assay CV < 7.2% (R&D Systems, Minneapolis, MN).

Tail vein blood samples were allowed to clot on ice and then centrifuged for 10 min at 10,000 × g. Serum samples were stored at −80°C until analyzed. Serum and liver triglycerides (TG) and cholesterol were measured according to manufacturer’s instructions using an Infinity kit (Thermo Fisher Scientific, Middletown, VA). Non-esterified free fatty acids (NEFA) were measured using a Wako kit (Wako Chemicals, Richmond, VA). Serum β-hydroxybutyrate (ketone) concentrations were measured with a StanBio Liquicolor kit (StanBio Laboratory, Boerne, TX). Serum insulin (Crystal Chem, Elk Grove Village, IL; cat # 90080), testosterone (ALPCO, Salem, NH; cat# 55-TESMS-E01), IL-1β, IL-6, TNF-α, MCP-1/CCL2 (all from R&D Systems, Minneapolis, MN; cat # DY401-05, DY406-05, DY410-05, DY479-05) levels were measured by ELISA according to manufacturer’s instructions.