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Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy.

Lorin S, Tol MJ, Bauvy C, Strijland A, Poüs C, Verhoeven AJ, Codogno P, Meijer AJ - Autophagy (2013)

Bottom Line: Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling.Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy.The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: EA4530, Faculty of Pharmacy, University Paris-Sud, Châtenay-Malabry, France.

ABSTRACT
Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

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Figure 2. Leucine, not valine, inhibits autophagy. (A) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells cultured in complete medium or EBSS supplemented with 1, 5 or 10 mM of leucine or valine for 4 h. Immunoblotting of ACTB was used as a loading control. The LC3-II/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (B) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells cultured in complete medium or EBSS supplemented with 10 mM of leucine or valine for 4 h. The number of GFP-LC3 dots per cell was scored on 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05.
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Figure 2: Figure 2. Leucine, not valine, inhibits autophagy. (A) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells cultured in complete medium or EBSS supplemented with 1, 5 or 10 mM of leucine or valine for 4 h. Immunoblotting of ACTB was used as a loading control. The LC3-II/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (B) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells cultured in complete medium or EBSS supplemented with 10 mM of leucine or valine for 4 h. The number of GFP-LC3 dots per cell was scored on 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05.

Mentions: The activity of GLUD1 is specifically regulated by the amino acid leucine, which has been extensively studied as a potent suppressor of autophagy in many different cell types. In the first series of experiments, we confirmed the inhibitory effect of leucine on starvation-induced autophagy (Fig. 2). Leucine inhibited accumulation of LC3-II in a concentration-dependent manner upon nutrient starvation (Fig. 2A). Similarly, leucine prevented the accumulation of GFP-LC3 punctate structures (Fig. 2B). To verify that the inhibitory effect of leucine on autophagy was specific, we repeated the starvation experiments in the presence of the amino acid valine. The inhibitory effect of leucine on autophagy upon nutrient starvation could not be recapitulated by valine, as judged by the degree of LC3-II accumulation (Fig. 2A) and of GFP-LC3 puncta (Fig. 2B). From these results, we concluded that leucine indeed strongly inhibits starvation-induced formation of autophagosomes.


Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy.

Lorin S, Tol MJ, Bauvy C, Strijland A, Poüs C, Verhoeven AJ, Codogno P, Meijer AJ - Autophagy (2013)

Figure 2. Leucine, not valine, inhibits autophagy. (A) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells cultured in complete medium or EBSS supplemented with 1, 5 or 10 mM of leucine or valine for 4 h. Immunoblotting of ACTB was used as a loading control. The LC3-II/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (B) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells cultured in complete medium or EBSS supplemented with 10 mM of leucine or valine for 4 h. The number of GFP-LC3 dots per cell was scored on 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3672295&req=5

Figure 2: Figure 2. Leucine, not valine, inhibits autophagy. (A) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells cultured in complete medium or EBSS supplemented with 1, 5 or 10 mM of leucine or valine for 4 h. Immunoblotting of ACTB was used as a loading control. The LC3-II/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (B) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells cultured in complete medium or EBSS supplemented with 10 mM of leucine or valine for 4 h. The number of GFP-LC3 dots per cell was scored on 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05.
Mentions: The activity of GLUD1 is specifically regulated by the amino acid leucine, which has been extensively studied as a potent suppressor of autophagy in many different cell types. In the first series of experiments, we confirmed the inhibitory effect of leucine on starvation-induced autophagy (Fig. 2). Leucine inhibited accumulation of LC3-II in a concentration-dependent manner upon nutrient starvation (Fig. 2A). Similarly, leucine prevented the accumulation of GFP-LC3 punctate structures (Fig. 2B). To verify that the inhibitory effect of leucine on autophagy was specific, we repeated the starvation experiments in the presence of the amino acid valine. The inhibitory effect of leucine on autophagy upon nutrient starvation could not be recapitulated by valine, as judged by the degree of LC3-II accumulation (Fig. 2A) and of GFP-LC3 puncta (Fig. 2B). From these results, we concluded that leucine indeed strongly inhibits starvation-induced formation of autophagosomes.

Bottom Line: Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling.Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy.The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: EA4530, Faculty of Pharmacy, University Paris-Sud, Châtenay-Malabry, France.

ABSTRACT
Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

Show MeSH
Related in: MedlinePlus