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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 1. Knockdown of GLUD1 stimulates autophagy and inhibits MTORC1 activity. (A) Immunoblot analysis of GLUD1, LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. The cells were cultured in complete medium. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h to block the lysosomal degradation of LC3-II. 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) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. Cells were cultured in complete medium. Immunoblotting of ACTB was used as a loading control. The SQSTM1/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (C) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells following a 72 h transfection with control (ct) siRNA or GLUD1 siRNA. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h. The number of GFP-LC3 dots per cell was scored on 50 to 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05. (D) Immunoblot analysis of phospho-RPS6 (P-RPS6), RPS6, phospho-EIF4EBP1 (P-EIF4EBP1) and EIF4EBP in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA, cultured in complete medium. Columns: mean; bars: SEM (n = 3); *p < 0.05.
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Figure 1: Figure 1. Knockdown of GLUD1 stimulates autophagy and inhibits MTORC1 activity. (A) Immunoblot analysis of GLUD1, LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. The cells were cultured in complete medium. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h to block the lysosomal degradation of LC3-II. 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) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. Cells were cultured in complete medium. Immunoblotting of ACTB was used as a loading control. The SQSTM1/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (C) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells following a 72 h transfection with control (ct) siRNA or GLUD1 siRNA. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h. The number of GFP-LC3 dots per cell was scored on 50 to 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05. (D) Immunoblot analysis of phospho-RPS6 (P-RPS6), RPS6, phospho-EIF4EBP1 (P-EIF4EBP1) and EIF4EBP in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA, cultured in complete medium. Columns: mean; bars: SEM (n = 3); *p < 0.05.

Mentions: To evaluate a potential role for GLUD1 in the regulation of autophagy, we employed an RNA interference approach in HeLa cells, resulting in 80% to 90% reduction of GLUD1 protein (Fig. 1A). We then assessed the effect of GLUD1 knockdown on the conversion of cytosolic LC3-I to the lipid-bound LC3-II upon autophagosome formation. The amount of LC3-II reflects the number of autophagosomes and is widely accepted as a reliable measurement of autophagic flux.32 Because the steady-state level of autophagosomes is dependent on both “de novo” synthesis and consumption in the lysosomal compartment, the effects on autophagic flux are best determined in the presence of bafilomycin A1, which blocks the fusion and subsequent content degradation of autophagosomes in the lysosome. Silencing GLUD1 led to a significant increase of LC3-II, both in the presence and absence of bafilomycin A1 (Fig. 1A). In agreement, we observed diminished levels of the autophagy cargo protein SQSTM1/p62 in GLUD1-depleted cells, indicative of increased autophagic degradation (Fig. 1B). We next evaluated the effect of GLUD1 depletion on cellular LC3 distribution in HeLa cells stably transfected with GFP-LC3. In line with the results obtained with western blot analysis, GLUD1 knockdown led to a marked increase in GFP-positive punctate structures compared with control cells (Fig. 1C), indicating that GLUD1 is involved in the regulation of autophagy.


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 1. Knockdown of GLUD1 stimulates autophagy and inhibits MTORC1 activity. (A) Immunoblot analysis of GLUD1, LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. The cells were cultured in complete medium. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h to block the lysosomal degradation of LC3-II. 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) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. Cells were cultured in complete medium. Immunoblotting of ACTB was used as a loading control. The SQSTM1/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (C) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells following a 72 h transfection with control (ct) siRNA or GLUD1 siRNA. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h. The number of GFP-LC3 dots per cell was scored on 50 to 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05. (D) Immunoblot analysis of phospho-RPS6 (P-RPS6), RPS6, phospho-EIF4EBP1 (P-EIF4EBP1) and EIF4EBP in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA, cultured in complete medium. Columns: mean; bars: SEM (n = 3); *p < 0.05.
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Figure 1: Figure 1. Knockdown of GLUD1 stimulates autophagy and inhibits MTORC1 activity. (A) Immunoblot analysis of GLUD1, LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. The cells were cultured in complete medium. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h to block the lysosomal degradation of LC3-II. 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) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA. Cells were cultured in complete medium. Immunoblotting of ACTB was used as a loading control. The SQSTM1/ACTB ratio was determined using Bio-1D quantification software. Columns: mean; bars: SEM (n = 3); *p < 0.05. (C) Representative images of GFP-LC3 staining in HeLa GFP-LC3 cells following a 72 h transfection with control (ct) siRNA or GLUD1 siRNA. Where indicated, 200 nM of bafilomycin A1 (BAF) was present for 2 h. The number of GFP-LC3 dots per cell was scored on 50 to 100 cells. Columns: mean; bars: SEM (n = 3); *p < 0.05. (D) Immunoblot analysis of phospho-RPS6 (P-RPS6), RPS6, phospho-EIF4EBP1 (P-EIF4EBP1) and EIF4EBP in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA, cultured in complete medium. Columns: mean; bars: SEM (n = 3); *p < 0.05.
Mentions: To evaluate a potential role for GLUD1 in the regulation of autophagy, we employed an RNA interference approach in HeLa cells, resulting in 80% to 90% reduction of GLUD1 protein (Fig. 1A). We then assessed the effect of GLUD1 knockdown on the conversion of cytosolic LC3-I to the lipid-bound LC3-II upon autophagosome formation. The amount of LC3-II reflects the number of autophagosomes and is widely accepted as a reliable measurement of autophagic flux.32 Because the steady-state level of autophagosomes is dependent on both “de novo” synthesis and consumption in the lysosomal compartment, the effects on autophagic flux are best determined in the presence of bafilomycin A1, which blocks the fusion and subsequent content degradation of autophagosomes in the lysosome. Silencing GLUD1 led to a significant increase of LC3-II, both in the presence and absence of bafilomycin A1 (Fig. 1A). In agreement, we observed diminished levels of the autophagy cargo protein SQSTM1/p62 in GLUD1-depleted cells, indicative of increased autophagic degradation (Fig. 1B). We next evaluated the effect of GLUD1 depletion on cellular LC3 distribution in HeLa cells stably transfected with GFP-LC3. In line with the results obtained with western blot analysis, GLUD1 knockdown led to a marked increase in GFP-positive punctate structures compared with control cells (Fig. 1C), indicating that GLUD1 is involved in the regulation of autophagy.

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