Soluble RANKL exaggerates hindlimb suspension-induced osteopeniabut not muscle protein balance

We examined the hypothesis that exaggerating unloading-induced bone loss using a combination of hindlimb suspension (HLS) and exogenous injections of receptor activator of nuclear factor kappa-B ligand (RANKL) also exaggerates muscle loss. Forty, male C57Bl/6J mice (16 weeks) were subjected to HLS or normal ambulation (ground control, GC) for 14 days. Mice received 3 intraperitoneal injections of either human recombinant soluble RANKL or PBS as control (n=10/group) at 24 hour intervals starting on Day 1 of HLS. GC + RANKL and HLS mice exhibited similar decreases in trabecular bone volume and density in both proximal tibias and distal femurs. However, RANKL affected trabecular number, separation, and connectivity density, while HLS decreased trabecular thickness. The combination of RANKL and HLS exacerbated these changes. Similarly, GC + RANKL and HLS mice saw comparable decreases in cortical bone volume, thickness, and strength in femur midshafts, and combination treatment exacerbated these changes. Plasma concentrations of P1NP were increased in both groups receiving RANKL, while CTX concentrations were unchanged. HLS decreased gastrocnemius weight and was associated with a reduction in global protein synthesis, and no change in proteasome activity. This change was correlated with a decrease in S6K1 and S6 phosphorylation, but no change in 4E-BP1 phosphorylation. Injection of RANKL did not alter muscle protein metabolism in GC or HLS mice. Our results suggest that injection of soluble RANKL exacerbates unloading-induced bone loss, but not unloading-induced muscle loss. This implies a temporal disconnect between muscle and bone loss in response to unloading.