Abstract
ABSTRACTAutophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.
Highlights
Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly
We have previously shown how amino acid starvation robustly activated autophagy in mouse embryonic fibroblasts (MEF) and how this response was fully blocked upon ULK1/2 DKO [11]
Glucose starvation led to only relatively small increases in LC3-II that did not further accumulate when lysosomal activity was blocked by bafilomycin (Baf) A1, which clearly contrasted with our previous observations following amino acid starvation using the same cell system [11]
Summary
Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). Mechanistic target of rapamycin complex 1 (MTORC1) phosphorylates ULK1 on Ser757 (Ser758 in humans), which has the effect of disrupting interaction between ULK1 and AMP-activated protein kinase (AMPK) [5] This direct binding allows AMPK to phosphorylate ULK1 on sites Ser317 and Ser777, which stimulates ULK1 activity for autophagy. The MTORC1-AMPK-ULK1 interplay model predicts that autophagy following amino acid withdrawal still requires AMPK function. Regarding this issue, the precise roles of glucose starvation and AMPK in autophagy remain controversial. Other reports have shown glucose starvation to inhibit autophagy responses [22, 23, 24]
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