Abstract
Propionic acidemia (PA) is an inborn error of metabolism caused by propionyl-CoA carboxylase (PCC) deficiency due to mutations in either PCCA or PCCB . Without proper management, the disease is associated with high mortality. Even with dietary restriction, patients often develop complications later in life, and the underlying pathological mechanisms remain poorly understood. The liver is the primary organ responsible for propionyl-CoA metabolism, yet the metabolic alterations induced by PCC deficiency in the liver have not been systematically investigated. In this study, we used a hepatocyte model of PA- PCCA (null) - HepG2 cells-to comprehensively examine metabolic alterations using stable isotope-based metabolic flux analysis. The PCCA knockout recapitulated key metabolic features of PA in HepG2 cells. Furthermore, PCCA deficiency reduced mitochondrial fatty acid oxidation while increasing glucose oxidation through pyruvate dehydrogenase. In contrast, pyruvate anaplerosis via pyruvate carboxylase was markedly reduced in PCCA knockout cells. This reduction in anaplerotic flux impaired the capacity for gluconeogenesis and lipid synthesis, consistent with observations from in vivo studies in Pcca⁻/⁻ (A138T) mice. Additionally, branched-chain keto acid catabolism was reduced in PCCA knockout HepG2 cells. Threonine showed minimal metabolic contribution in this model, further supporting the role of propionate as a major source of propionyl-CoA production. Collectively, these findings highlight the metabolic vulnerabilities associated with PCC deficiency and underscore the increased risk of prolonged fasting in patients with PA, particularly those with severe disease.