Glutamine energy substrate anaplerosis increases bone density in the Pah enu2 classical PKU mouse in the absence of phenylalanine restriction.

Osteopenia is an under-investigated clinical presentation of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU). While osteopenia is not fully penetrant in human PKU, the Pah enu2 mouse is universally osteopenic and ideal to study the phenotype. We determined Pah enu2 mesenchymal stem cells (MSCs) are developmentally impaired in the osteoblast lineage. Moreover, we determined energy dysregulation and oxidative stress contribute to the osteoblast developmental deficit. The MSC preferred substrate glutamine (Gln) was applied to enhance energy homeostasis. In vitro Pah enu2 MSCs, in the context of 1200 μM Phe, respond to Gln with increased in situ alkaline phosphatase activity indicating augmented osteoblast differentiation. Oximetry applied to Pah enu2 MSCs in osteoblast differentiation show Gln energy substrate increases oxygen consumption, specifically maximum respiration and respiratory reserve. For 60 days post-weaning, Pah enu2 animals received either no intervention (standard lab chow), amino acid defined chow maintaining plasma Phe at ~200 μM, or standard lab chow where ad libitum water was a 2% Gln solution. Bone density was assessed by microcomputed tomography and bone growth assessed by dye labeling. Bone density and dye labeling in Phe-restricted Pah enu2 was indistinguishable from untreated Pah enu2 . Gln energy substrate provided to Pah enu2 , in the context of uncontrolled hyperphenylalaninemia, present increased bone density and dye labeling. These data provide further evidence that Pah enu2 MSCs experience a secondary energy deficit that is responsive both in vitro and in vivo to Gln energy substrate and independent of hyperphenylalaninemia. Energy support may have effect to treat human PKU osteopenia and elements of PKU neurologic disease resistant to standard of care systemic Phe reduction. Glutamine energy substrate anaplerosis increased Pah enu2 bone density and improved in vitro MSC function in the context of hyperphenylalaninemia in the classical PKU range.