Abstract
Pathophysiology of osteopenia in phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is poorly characterized. The Pahenu2 mouse is universally osteopenic where dietary phenylalanine (Phe) management with amino acid defined chow does not improve bone density. We previously demonstrated Pahenu2 osteopenia owes to a skeletal stem cell (SSC) developmental deficit mediated by energy dysregulation and oxidative stress. This investigation demonstrates complexity of Pahenu2 SSC energy dysregulation. Creatine use by bone tissue is recognized. In vitro Pahenu2 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 Pahenu2 and control cohorts. Control cohorts include unaffected littermates (wt/wt), Pahenu2 receiving no intervention, and dietary Phe restricted Pahenu2. Experimental cohorts (Phe unrestricted Pahenu2, Phe restricted Pahenu2) were provided 1% creatine ad libitum in water. After 60 days, microcomputed tomography assessed bone metrics. Equivalent osteopenia occurs in Phe-restricted and untreated Pahenu2 control cohorts. In Phe unrestricted Pahenu2, creatine was without effect as bone density remained equivalent to Pahenu2 control cohorts. Alternatively, Phe-restricted Pahenu2 receiving creatine present increased bone density. We hypothesize small molecule dysregulation in untreated Pahenu2 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.