Fig. 3.

Ammonia stimulates mTOR-independent autophagy. (A) Glucose deprivation, but not amino acid starvation, increases ammonia production. Ammonia levels in media of cells cultured for 24 h were determined with the Nova Biomedical Flex Analyzer. *P < 0.05. (B) NH₄Cl treatment increases autophagy without affecting mTOR activity. WT MEFs expressing GFP-LC3 were treated with varying concentrations of NH₄Cl in complete medium for 24 h. Autophagy was determined by GFP-LC3 processing and LC3 conversion. mTOR activity also was assessed by immunoblotting for phospho-p70 S6 kinase (pS6K). (C) Atg5, but not Ulk1/2, is required for ammonia-induced autophagy. The GFP-LC3–expressing WT, Ulk1/2 DKO (1/2^−/−), and Atg5 KO (5^−/−) MEFs were treated with 2 mM NH₄Cl for 24 h. Autophagy was measured by immunoblotting for GFP-LC3 processing. (D) The dose of NH₄Cl that induces autophagy has no effect on cell viability. After 48 h incubation at the conditions indicated in C, cell viability was determined by propidium iodide (PI) exclusion.