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. 2017 Aug 24;8(1):338.
doi: 10.1038/s41467-017-00369-y.

Glutamine metabolism regulates autophagy-dependent mTORC1 reactivation during amino acid starvation

Hayden Weng Siong Tan  1 , Arthur Yi Loong Sim  1 , Yun Chau Long  2
Affiliations

Glutamine metabolism regulates autophagy-dependent mTORC1 reactivation during amino acid starvation

Hayden Weng Siong Tan et al. Nat Commun. .

Abstract

Activation of autophagy and elevation of glutamine synthesis represent key adaptations to maintain amino acid balance during starvation. In this study, we investigate the role of autophagy and glutamine on the regulation of mTORC1, a critical kinase that regulates cell growth and proliferation. We report that supplementation of glutamine alone is sufficient to restore mTORC1 activity during prolonged amino acid starvation. Inhibition of autophagy abolishes the restorative effect of glutamine, suggesting that reactivation of mTORC1 is autophagy-dependent. Inhibition of glutaminolysis or transamination impairs glutamine-mediated mTORC1 reactivation, suggesting glutamine reactivates mTORC1 specifically through its conversion to glutamate and restoration of non-essential amino acid pool. Despite a persistent drop in essential amino acid pool during amino acid starvation, crosstalk between glutamine and autophagy is sufficient to restore insulin sensitivity of mTORC1. Thus, glutamine metabolism and autophagy constitute a specific metabolic program which restores mTORC1 activity during amino acid starvation.mTORC1 is a critical kinase that regulates cell growth and proliferation. Here the authors show that glutamine metabolism is sufficient to restore mTORC1 activity during prolonged amino acid starvation in an autophagy-dependent manner.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Glutamine reactivates mTORC1 signaling during amino acid starvation. a Wild-type MEFs were starved of amino acids with or without the supplementation of glutamine (4 mM) for the indicated durations. Changes in mTORC1 signaling were assessed by immunoblotting of p-4EBP1, p-ULK1, and p-p70S6K. b Wild-type MEFs were deprived of amino acids with the supplementation of glutamine (4 mM) for the indicated durations. Torin 1 (250 nM) was added upon induction of amino acid starvation to inhibit mTORC1. c Wild-type MEFs were deprived of amino acids for 5 h and cells were treated with H2O (vehicle add-back) or 4 mM glutamine (Gln add-back) for the indicated durations. d HepG2 cells were subjected to amino acid starvation in the absence or presence of glutamine (4 mM) for the indicated durations. e HepG2 cells were starved of amino acids with the supplementation of glutamine (4 mM) for the indicated durations. Rapamycin (50 nM) or Torin 1 (250 nM) was added upon induction of starvation to inhibit mTORC1. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1. f Wild-type MEFs were starved of amino acids with or without the supplementation of glutamine (4 mM) for 1 or 5 h. Cells were then fixed and co-stained with antibodies specific for mTOR (green) and the lysosomal marker Lamp2a (red). The scale bar represents 10 µm
Fig. 2
Fig. 2
Autophagy is essential for glutamine-dependent restoration of mTORC1 signaling. a Wild-type (Atg5+/+) or Atg5-knockout (Atg5−/−) MEFs were subjected to amino acid starvation in the presence of glutamine (4 mM) for the indicated durations. b Wild-type MEFs were deprived of amino acids with the supplementation of glutamine (4 mM) for the indicated durations. Bafilomycin A1 (Baf, 200 nM), concanamycin A (ConA, 100 nM) or SAR405 (SAR, 5 µM) was added to inhibit autophagy. Changes in mTORC1 activity or autophagy were assessed by immunoblotting of p-4EBP1 and p-ULK1, or LC3, respectively. c, d HepG2 cells and C2C12 mouse myotubes were starved of amino acids as in b for the indicated durations. SAR405 (5 µM) and bafilomycin A1 (200 nM) was added to inhibit autophagy in HepG2 cells and C2C12 myotubes, respectively. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1 (for HepG2 cells) or p-p70S6K (for C2C12 myotubes), and changes in autophagy was evaluated by immunoblotting of LC3. e Wild-type MEFs were deprived of amino acids with the supplementation of glutamine (4 mM) for the indicated durations. MG132 (10 µM) was added to inhibit proteasome function. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1
Fig. 3
Fig. 3
Glutamine metabolism is essential for mTORC1 reactivation during amino acid starvation. a Wild-type MEFs were starved of amino acids with (black line) or without (gray line) the supplementation of glutamine (4 mM) for the indicated durations. Individual non-essential amino acid (NEAA) levels were measured from cell lysates by GC-TOF-MS. Data are expressed as the fold change of control cells (unstarved control, 0 h). Data are the mean ± SEM of n = 4–5, *P≤0.05; **P≤0.01 (between the groups with and without glutamine supplementation at the same time point via Student’s t-test). b Wild-type MEFs or c HepG2 cells were deprived of amino acids with the supplementation of glutamine (4 mM) for the indicated durations in the absence or presence of BPTES (10 µM). Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1. d Schematic diagram of glutamine metabolism. Glutamine is converted to glutamate by glutaminase, which is inhibited by BPTES. Glutamate undergoes transamination (which is inhibited by amino-oxyacetate, AOA, or knockdown of GOT2/GPT2), to produce non-essential amino acids (NEAA) and α-KG (a TCA cycle intermediate). e Wild-type MEFs were starved of amino acids with the supplementation of 4 mM glutamine, glutamate or alanine in the presence or absence of AOA (5 mM). f Wild-type MEFs were subjected to siRNA-mediated knockdown of GOT2 and GPT2, and cells were deprived of amino acids with the supplementation of glutamine (4 mM) for the indicated durations. g Wild-type MEFs were starved of amino acids with or without the supplementation of NEAA for the indicated durations. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1
Fig. 4
Fig. 4
Glutamine reactivates mTORC1 signaling independent of EAAs. a, b Wild-type MEFs were starved of amino acids with (black line) or without (gray line) the supplementation of glutamine (4 mM) for the indicated durations. Individual essential amino acid a and TCA cycle intermediate b levels were measured from cell lysates by GC-TOF-MS. Data are expressed as the fold change of control cells (unstarved control, 0 h). Data are the mean ± SEM of n = 4–5, *P≤0.05; **P≤0.01 (between the groups with and without glutamine supplementation at the same time point via Student’s t-test). c Wild-type MEFs were subjected to amino acid starvation with the supplementation of 4 mM glutamine, dimethyl-α-KG, malate or OAA for the indicated durations. d Wild-type MEFs were starved of amino acids with the supplementation of glutamine (4 mM) or tryptophan (4 mM) for the indicated durations. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1
Fig. 5
Fig. 5
Glutamine and autophagy sustain insulin-induced mTORC1 signaling during amino acid starvation. a HepG2 cells were pre-incubated for 30 min in EBSS with amino acids. Cells were then starved of amino acids in the presence or absence of glutamine (4 mM) with or without SAR405 (5 µM) for 5 h, followed by insulin (50 nM) stimulation for an additional 30 min. Changes in mTORC1 activity were assessed by immunoblotting of p-4EBP1 and p-ULK1. Insulin stimulation was assessed by immunoblotting of p-Akt. b C2C12 myotubes were pre-incubated for 30 min in EBSS with amino acids. Myotubes were then starved of amino acids in the presence or absence of glutamine (4 mM) with or without bafilomycin A1 (200 nM) for 5 h, followed by insulin (50 nM) stimulation for an additional 30 min. Changes in mTORC1 activity were assessed by immunoblotting of p-p70S6K. Insulin stimulation was assessed by immunoblotting of p-Akt
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