Fig. 4.

Tyr metabolism provides mitochondrial acetyl-CoA for oxidative phosphorylation. (A) Acetyl-CoA from ¹³C-glucose, ¹³C-glutamine, or ¹³C-Tyr in CD13⁺ and CD13^- fractions sorted from HepG2 and HuH7 cells. (B) The intracellular distribution of ¹³C-acetyl-CoA in HepG2-derived CD13⁺ cells incubated with ¹³C-tyrosine for 24 hours. (C) Upper panel: oxygen consumption of HepG2 and HuH7-derived CD13⁺ and CD13^- cells cultured in complete or Tyr-deprived medium for 24 hours; lower panel: apoptotic rate of HepG2 and HuH7-derived CD13⁺ and CD13^- cells cultured with oligomycin (2.5 μg/mL) for 72 hours. (D) Oxygen consumption of HepG2 and HuH7-derived CD13⁺ cells cultured in Tyr-deprived medium and pretreated with 200 μM acetyl-CoA, 50 μM dimethyl-fumarate (DMF), or both for 24 hours. (E) HepG2-xenografted BALB/c nude mice were randomized to receive ¹³C-glucose (n=5), ¹³C-glutamine (n=5), or ¹³C-tyrosine (n=6) infusion for 200 minutes prior to cytometric sorting of CD13⁺ cells. The percentage of ¹³C-labeled TCA cycle intermediates was assessed by mass spectrometry. (F) Plots of oxygen consumption rate as a parameter of time in the absence or presence of 100 nM Nitisinone (indicated by arrow) in CD13⁺ fraction sorted from HuH7 cells. (G) ATP levels in HepG2 and HuH7-derived CD13⁺ fraction treated with or without nitisinone (100 nM) for 6 hours. (H) Mitochondrial reactive oxygen species (ROS; left panel) and reduced glutathione (GSH) levels (right panel) in HepG2 and HuH7-derived CD13⁺ cells subjected to 100 nM nitisinone for 6 hours. Values shown are mean±standard deviation. A two-tailed unpaired t test was used to compare experimental groups. *p < 0.05.