Recent studies have demonstrated that erythropoietin (EPO) treatment in mice results in trabecular bone loss. Here, we investigated the dose-response relationship between EPO, hemoglobin (Hgb) and bone loss and examined the reversibility of EPO-induced damage. Increasing doses of EPO over two weeks led to a dose-dependent increase in Hgb in young female mice, accompanied by a disproportionate decrease in trabecular bone mass measured by micro-CT (µCT). Namely, increasing EPO from 24 to 540 IU/week produced a modest 12% rise in Hgb (20.2 ± 1.3 mg/dL vs 22.7 ± 1.3 mg/dL), while trabecular bone volume fraction (BV/TV) in the distal femur decreased dramatically (27 ± 8.5% vs 53 ± 10.2% bone loss). To explore the long-term skeletal effects of EPO, we treated mice for two weeks (540 IU/week) and monitored bone mass changes after treatment cessation. Six weeks post-treatment, there was only a partial recovery of the trabecular microarchitecture in the femur and vertebra. EPO-induced bone loss is therefore dose-dependent and mostly irreversible at doses that offer only a minor advantage in the treatment of anemia. Because patients requiring EPO therapy are often prone to osteoporosis, our data advocate for using the lowest effective EPO dose for the shortest period of time to decrease thromboembolic complications and minimize the adverse skeletal outcome.

Palmitic acid (PA) is the most abundant saturated fatty acid in human milk, where it is heavily concentrated in the sn-2-position (termed beta palmitate, BPA) and as such is conserved in all women, regardless of their diet or ethnicity, indicating its physiological and metabolic importance. We hypothesized that BPA improves the efficiency of nutrition-induced catch up growth as compared to sn-1,3 PA, which is present in vegetable oil. Pre-pubertal male rats were subjected to a 17 days food restriction followed by re-feeding for nine days with 1,3 PA or BPA-containing diets. We measured bone length, epiphyseal growth plate height (EGP, histology), bone quality (micro-CT and 3-point bending assay), and gene expression (Affymetrix). The BPA-containing diet improved most growth parameters: humeri length and EGP height were greater in the BPA-fed animals. Further analysis of the EGP revealed that the hypertrophic zone was significantly higher in the BPA group. In addition, Affymetrix analysis revealed that the diet affected the expression of several genes in the liver and EGP. Despite the very subtle difference between the diets and the short re-feeding period, we found a small but significant improvement in most growth parameters in the BPA-fed rats. This pre-clinical study may have important implications, especially for children with growth disorders and children with special nutritional needs.

The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin⁻CD11b⁻Ly6C^hi CD115⁺, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment.