Recombinant irisin

Eighteen Sprague-Dawley rats (male, 6 weeks old) were ordered from Envigo (Houston, TX) and singly housed in an institutionally approved animal facility with 12-h light–dark cycles. At 7 weeks of age, rodents were switched from the standard rodent chow of the animal facility (Teklad 2018; Envigo, Houston, TX, USA) to the purified AIN93G chow for growing rodents (Research Diets, Inc., New Brunswick, NJ, USA). The age of animals and the diet were utilized to compare to previous work with age-matched and diet-matched groups^{51 (link),52 (link)}. At 8 weeks of age, animals were randomly divided into three different groups (n = 6/group): ambulatory cage control (Con), hindlimb unloaded (HU), and hindlimb unloaded with exogenous irisin treatment (HU + Ir). The irisin-treated HU group was given injections of recombinant irisin (Adipogen, San Diego, CA, USA) intraperitoneally 3×/week as previously described^{51 (link),52 (link)}. HU was maintained continuously for 28 days. Fluorochrome calcein labels (Sigma Aldrich, St Louis, MO, USA) were injected intraperitoneally 8 and 3 days prior to termination to label mineralized surfaces on bone. Following 4 weeks of HU, animals were euthanized and tissues were collected. All animal procedures were approved by the Texas A&M Institutional Animal Use and Care Committee and conform to the NIH Guide for the Care and Use of Laboratory Animals.

Mice were subcutaneously injected with Recombinant irisin at 100 µg/kg (n = 12) or vehicle (0.9% saline solution sterilized by 0.22 μ filtration) (n = 12) once a day for a total of five consecutive days. Recombinant irisin was provided by Adipogen International (San Diego, CA, USA). Treatment was regularly executed at the same time each day. The concentration of irisin was chosen on the basis of previous studies performed by our research team [6 (link),24 (link)].