Concentrations of adrenocorticotropic hormone too low to effect cortisol enhance osteogenesis in vitro and in vivo

After finding that minimal amounts of adrenocorticotropic hormone (ACTH_1-24) prevented osteonecrosis in rabbits, we studied bone formation at nanomolar ACTH_1-24, in vivo in rabbits and in vitro in human osteoblasts. ACTH_1-24 in rabbits at 0.6 μg/kg/day had no measurable effect on cortisol. Groups of five rabbits given 0.6 μg/kg/day of ACTH_1-24 enhanced trabecular bone in the rabbit femoral head relative to saline-treated controls (controls), increased bone volume/total volume (BV/TV) by micro-computed tomography, <i>P</i> < 0.03. Xylenol orange and calcein labeling in vivo showed increased trabecular bone formation with 0.6 μg/kg/day of ACTH_1-24, <i>P</i> = 0.0089 vs. controls. In contrast, the cortex of the femoral shaft was unaffected, BV/TV, <i>P</i> > 0.95 ACTH_1-24 vs. controls. Bone marrow mRNA by PCR showed no change in osteoclast markers and confirmed increased osteoblast markers, <i>P</i> < 0.05. In vitro, ACTH_1-24 elevated expression of collagen 1, alkaline phosphatase (ALP), osteocalcin (bone gamma carboxyglutamate protein), and RunX2 in human osteoblasts differentiated on polyethylene terephthalate (PET) membranes. Optimal response was at 10^-9 to 10^-12 M. Vascular endothelial growth factor (VEGF) receptors fms-related receptor tyrosine kinase 1 and FLK-1, and ACTH_1-24 receptors MC2R were upregulated at 10^-12 M ACTH_1-24. Pathway analysis included increased bone morphogenetic protein 2, Smad1, Wnt-1, β-catenin, and transforming growth factor, beta 1 pathways. Because bone-forming osteoblasts are metabolically highly active, we studied mRNA expression of mitochondrial complex 1 (NDUFA5, NDUFS2, NDUFB1, and NDUFB6) members with key roles in energy production. This increased at 10^-12 M ACTH_1-24. An ELISA for mitochondrial complex 1 activity showed maximum activity at 10^-9 M and high activity at 10^-12 M ACTH_1-24. Thus, long-term very low-dose ACTH_1-24 increases bone formation in vivo and in vitro.<b>NEW & NOTEWORTHY</b> This is the first study to assess directly the effects of very low concentrations of ACTH_1-24, picomolar to micromolar, on regulation of bone growth. It shows effects on bone mass in the femoral heads and by labeling bone formation in rabbits in vitro, and effect on key proteins related to bone growth mechanisms in cell preparations of human bone-forming cells in vitro.