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FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1.

ABSTRACT: The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle.

SUBMITTER: Wu X

PROVIDER: S-EPMC4900541 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1.

Wu Xiaochun X Zhao Lingling L Chen Zhi Z Ji Xin X Qiao Xianfeng X Jin Yaping Y Liu Wei W

PloS one 20160609 6

The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by ...[more]

PMID: 27280402

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Project description:ObjectiveExercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown.MethodsC57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of l-leucine or saline acutely or 48 h after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[14C(U)]-leucine tracer labeling.ResultsWhen compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise.ConclusionsThe enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestion is not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

Dataset Information

FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1.

Publications

FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1.

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