Autophagy Regulates Foxo1 Protein Abundance to Control Hepatic Glucose Production in Glucagon Signalling

AIM: During fasting, the liver undergoes coordinated metabolic adaptations to maintain systemic energy homeostasis. These adaptations are driven in part by glucagon, which promotes hepatic glucose production (HGP) and fatty acid oxidation (FAO) through mechanisms involving the transcription factor Foxo1. Autophagy is also activated during nutrient deprivation and maintains intracellular nutrient availability by recycling cellular components. However, whether autophagy supports glucagon-Foxo1 signalling and the associated hepatic metabolic responses remains unclear. MATERIALS AND METHODS: Hydroxychloroquine sulphate (HCQ) and AAV8-shRNA-Atg7 were used to inhibit autophagy in control and liver-specific Foxo1 knockout (L-FKO) mice, and chloroquine (CQ) and siRNA-Atg7 were used to inhibit autophagy in mouse primary hepatocytes. Pyruvate and glucagon tolerance tests were performed in mice. HGP assays, gene expression analysis, western blotting and HPLC-based amino acid measurements were conducted to evaluate glucose production, Foxo1 signalling, gluconeogenic and FAO-related pathways and amino acid availability. RESULTS: We found that inhibition of autophagy reduced pyruvate-stimulated glucose excursions in mice and glucose production in isolated primary hepatocytes, while showing a trend towards reducing glucagon-stimulated glucose excursions in vivo. It also attenuated glucagon-responsive gluconeogenic and FAO-related gene expression in primary hepatocytes. However, these effects of autophagy inhibition were markedly blunted in Foxo1-deficient models. Mechanistically, suppression of autophagy significantly decreased Foxo1 protein abundance in association with impaired hepatic amino acid homeostasis, without corresponding changes in Foxo1 gene expression and not fully explained by proteasome-dependent degradation. Importantly, supplementation with exogenous amino acids restored Foxo1 protein levels by 1.89-fold under basal conditions and 1.35-fold under glucagon-treated conditions (p < 0.05), and partially rescued HGP under autophagy-inhibited conditions. CONCLUSION: These findings identify autophagy-regulated amino acid homeostasis as a critical mechanism that sustains Foxo1 protein abundance and supports glucagon-responsive hepatic metabolic adaptation during fasting.