AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK)1, but it has been unclear whether this occurs solely via changes in AMP or ADP, the classical activators of AMPK2–5. Here, we uncover a mechanism that triggers AMPK activation via an AMP/ADP-independent mechanism sensing absence of FBP, with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of lysosomal complexes containing the v-ATPase, Ragulator, AXIN, LKB1 and AMPK, previously shown to be required for AMPK activation6,7. Knockdown of aldolases activates AMPK even in cells with abundant glucose, while the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts association of AXIN/LKB1 with v-ATPase/Ragulator. Importantly, in some cell types AMP:ATP/ADP:ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.

In metazoans, cells depend on extracellular growth factors for energy homeostasis. We found that glycogen synthase kinase-3 (GSK3), when deinhibited by default in cells deprived of growth factors, activates acetyltransferase TIP60 through phosphorylating TIP60-Ser(86), which directly acetylates and stimulates the protein kinase ULK1, which is required for autophagy. Cells engineered to express TIP60(S86A) that cannot be phosphorylated by GSK3 could not undergo serum deprivation-induced autophagy. An acetylation-defective mutant of ULK1 failed to rescue autophagy in ULK1(-/-) mouse embryonic fibroblasts. Cells used signaling from GSK3 to TIP60 and ULK1 to regulate autophagy when deprived of serum but not glucose. These findings uncover an activating pathway that integrates protein phosphorylation and acetylation to connect growth factor deprivation to autophagy.

Liver kinase B1 (LKB1) has important roles in governing energy homeostasis by regulating the activity of the energy sensor kinase AMP-activated protein kinase (AMPK). The regulation of LKB1 function, however, is still poorly understood. Here we demonstrate that the orphan nuclear receptor Nur77 binds and sequesters LKB1 in the nucleus, thereby attenuating AMPK activation. This Nur77 function is antagonized by the chemical compound ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), which interacts with Nur77 with high affinity and at specific sites. TMPA binding of Nur77 results in the release and shuttling of LKB1 to the cytoplasm to phosphorylate AMPKα. Moreover, TMPA effectively reduces blood glucose and alleviates insulin resistance in type II db/db and high-fat diet- and streptozotocin-induced diabetic mice but not in diabetic littermates with the Nur77 gene knocked out. This study attains a mechanistic understanding of the regulation of LKB1-AMPK axis and implicates Nur77 as a new and amenable target for the design and development of therapeutics to treat metabolic diseases.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.