NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes

Abstract

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-α-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) on urea synthesis was studied in isolated rat hepatocytes. α-Methylisocitrate substantially inhibited the rate of urea synthesis (35-84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). α-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamate, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of α-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via ANDP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, α-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors of ornithine-2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the α-methylisocitrate-insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by α-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by α-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or α-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.