FoxO transcription factors coordinate the urea cycle and gluconeogenesis by controlling Ass1.

Amino acid catabolism during fasting requires coordinated nitrogen disposal and glucose production, but the transcriptional logic linking the urea cycle to gluconeogenesis remains unclear. Forkhead box O (FoxO) transcription factors regulate fasting metabolism, and here we identify FoxOs as direct hepatic regulators of argininosuccinate synthase 1 (Ass1). Acute hepatic FoxO1/3a knockdown in fasted mice reduced Ass1 expression, lowered blood glucose, and altered urea-cycle amino acids, with decreased arginine and increased ornithine, even in Klf15-deficient livers. Ass1 silencing phenocopied these effects and impaired glucose production from pyruvate and lactate, whereas Ass1 overexpression restored glucose production in FoxO-inhibited hepatocytes. Mechanistically, we identified a functional FoxO-binding element within an upstream Ass1 enhancer and confirmed its fasting-inducible activity by reporter assays, electrophoretic mobility shift assays, in vivo imaging, and chromatin immunoprecipitation. These findings establish a KLF15-independent FoxO-Ass1 axis that coordinates ureagenesis with gluconeogenesis during fasting and supports hepatic metabolic adaptation to nutrient deprivation.