Creatine energy substrate increases bone density in the Pah(enu2) classical PKU mouse in the context of phenylalanine restriction

Pathophysiology of osteopenia in phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is poorly characterized. The Pah(enu2) mouse is universally osteopenic where dietary phenylalanine (Phe) management with amino acid defined chow does not improve bone density. We previously demonstrated Pah(enu2) osteopenia owes to a skeletal stem cell (SSC) developmental deficit mediated by energy dysregulation and oxidative stress. This investigation demonstrates complexity of Pah(enu2) SSC energy dysregulation. Creatine use by bone tissue is recognized. In vitro Pah(enu2) SSCs in osteoblast differentiation respond to creatine with increased in situ alkaline phosphatase activity and increased intracellular ATP content. Animal studies applied a 60-day creatine regimen to Pah(enu2) and control cohorts. Control cohorts include unaffected littermates (wt/wt), Pah(enu2) receiving no intervention, and dietary Phe restricted Pah(enu2). Experimental cohorts (Phe unrestricted Pah(enu2), Phe restricted Pah(enu2)) were provided 1% creatine ad libitum in water. After 60 days, microcomputed tomography assessed bone metrics. Equivalent osteopenia occurs in Phe-restricted and untreated Pah(enu2) control cohorts. In Phe unrestricted Pah(enu2), creatine was without effect as bone density remained equivalent to Pah(enu2) control cohorts. Alternatively, Phe-restricted Pah(enu2) receiving creatine present increased bone density. We hypothesize small molecule dysregulation in untreated Pah(enu2) disallows creatine utilization; therefore, osteopenia persisted. Dietary Phe restriction enables creatine utilization to enhance SSC osteoblast differentiation and improve in vivo bone density. PKU intervention singularly focused on Phe reduction enables residual disease including osteopenia and neurologic elements. Intervention concurrently addressing Phe homeostasis and energy dysregulation will improve disease elements refractory to standard of care Phe reduction mono-therapy.