Although it is well established that animals adapted to a high-protein, carbohydrate-free (HP) diet maintain glycemia through enhanced hepatic gluconeogenesis, the regulatory factors and molecular mechanisms underlying this adaptation remain incompletely understood. Given the chronically elevated glucagon levels observed in these animals, we hypothesized that the cAMP/PKA/CREB signaling pathway might contribute to the enhanced gluconeogenic capacity observed in HP-fed mice. Although CREB activity was transiently increased during early HP feeding, it became attenuated upon prolonged exposure. This attenuation correlated with elevated hepatic GRK2 content, likely driven by increased circulating branched-chain amino acids (BCAAs) and suppression of hepatic autophagy. Exploring alternative regulatory pathways, we identified impaired insulin signaling and reduced phosphorylation and acetylation of hepatic FoxO1 in HP-adapted mice, supporting a central role for FoxO1 in sustaining gluconeogenesis. Consistently, pharmacological inhibition of FoxO1 reduced hepatic gluconeogenesis and glycemia, and suppressed the liver expression of,, and, key transcriptional coactivators associated with long-term gluconeogenic regulation. Furthermore, we found that elevated corticosterone levels in HP-adapted animals were essential for maintaining hepatic gluconeogenesis and its fasting glycemia. Together, our findings reveal a shift in the regulatory landscape of hepatic gluconeogenesis during HP feeding, transitioning from early CREB activation to a sustained FoxO1-driven transcriptional program.The regulation of hepatic glucose production under a high-protein (HP) diet remains unclear. We show that gluconeogenesis in HP-fed mice is initially driven by CREB but shifts to FoxO1 dependence over time. Notably, FoxO1 is essential for maintaining gluconeogenesis and glycemia in HP-adapted animals. We also reveal a key role for corticosterone in preserving gluconeogenic capacity and fasting glycemia. These findings provide insights into hepatic metabolic adaptation and into molecular mechanisms governing glycemic homeostasis. Ppargc1a Nr4a1Hnf4a NEW & NOTEWORTHY
