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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 3. Knockdown of GLUD1 prevents the inhibition of autophagy by leucine. (A) The number of GFP-LC3 dots per cell was scored on 50 to 100 cells following a 72 h transfection with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and a 4 h treatment with complete medium (CM), starved medium (EBSS) and starved medium supplemented with 10 mM Leucine (EBSS+Leu). Columns: mean; bars: SEM (n = 3); **p < 0.01. (B) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA for 72 h and cultured in EBSS supplemented with 10 mM leucine or valine for 4 h. 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 < 0.01. (C) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and cultured for 4 h in complete medium or EBSS supplemented with 10 mM leucine. 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 < 0.01. (D) Measurement of the degradation of long-lived proteins degradation in HeLa cells transfected with control (ct) siRNA (upper western blot and histogram) or GLUD1 siRNA (lower western blot and histogram) for 72 h and cultured in complete medium or EBSS supplemented with 10 mM leucine or 10 mM 3-methyladenine (3-MA). Columns: mean; bars: SEM (n = 3); *p < 0.05.
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Figure 3: Figure 3. Knockdown of GLUD1 prevents the inhibition of autophagy by leucine. (A) The number of GFP-LC3 dots per cell was scored on 50 to 100 cells following a 72 h transfection with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and a 4 h treatment with complete medium (CM), starved medium (EBSS) and starved medium supplemented with 10 mM Leucine (EBSS+Leu). Columns: mean; bars: SEM (n = 3); **p < 0.01. (B) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA for 72 h and cultured in EBSS supplemented with 10 mM leucine or valine for 4 h. 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 < 0.01. (C) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and cultured for 4 h in complete medium or EBSS supplemented with 10 mM leucine. 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 < 0.01. (D) Measurement of the degradation of long-lived proteins degradation in HeLa cells transfected with control (ct) siRNA (upper western blot and histogram) or GLUD1 siRNA (lower western blot and histogram) for 72 h and cultured in complete medium or EBSS supplemented with 10 mM leucine or 10 mM 3-methyladenine (3-MA). Columns: mean; bars: SEM (n = 3); *p < 0.05.

Mentions: We next investigated whether the regulation of autophagy by leucine is dependent on the expression of GLUD1. For this purpose, we assessed the effect of leucine on autophagy in both control cells and in GLUD1-depleted cells (Fig. 3). Again, in nutrient-starved control cells, the presence of leucine was sufficient to inhibit the biosynthesis of autophagosomes as judged by the number of GFP-LC3 puncta (Fig. 3A). In agreement, leucine normalized the autophagic flux to the level observed in cells cultured in complete medium, as shown by the absence of LC3-II accumulation (Fig. 3B) and impaired degradation of SQSTM1 (Fig. 3C). Finally, leucine also prevented starvation-induced long-lived protein degradation (Fig. 3D). Importantly, we found that knockdown of GLUD1 abolished the inhibitory effect of leucine on the accumulation of GFP-LC3 puncta (Fig. 3A). The increase in autophagosomes correlated with a sustained autophagic flux and lysosomal activity as determined by the accumulation of LC3-II in bafilomycin A1-treated cells (Fig. 3B) and by the degradation of SQSTM1 (Fig. 3C), which could no longer be suppressed by leucine in GLUD1-depleted cells. Similarly, the rate of starvation-induced long-lived protein degradation in GLUD1-depleted cells was shown to be insensitive to leucine addition, but remained sensitive to 3-MA (Fig. 3D), an inhibitor of autophagosome formation by interfering with the activity of PIK3C3 (the class III PtdIns3K).33 Taken together, these results strongly suggest that GLUD1 is required for the inhibitory effect of leucine on 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 3. Knockdown of GLUD1 prevents the inhibition of autophagy by leucine. (A) The number of GFP-LC3 dots per cell was scored on 50 to 100 cells following a 72 h transfection with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and a 4 h treatment with complete medium (CM), starved medium (EBSS) and starved medium supplemented with 10 mM Leucine (EBSS+Leu). Columns: mean; bars: SEM (n = 3); **p < 0.01. (B) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA for 72 h and cultured in EBSS supplemented with 10 mM leucine or valine for 4 h. 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 < 0.01. (C) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and cultured for 4 h in complete medium or EBSS supplemented with 10 mM leucine. 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 < 0.01. (D) Measurement of the degradation of long-lived proteins degradation in HeLa cells transfected with control (ct) siRNA (upper western blot and histogram) or GLUD1 siRNA (lower western blot and histogram) for 72 h and cultured in complete medium or EBSS supplemented with 10 mM leucine or 10 mM 3-methyladenine (3-MA). Columns: mean; bars: SEM (n = 3); *p < 0.05.
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Figure 3: Figure 3. Knockdown of GLUD1 prevents the inhibition of autophagy by leucine. (A) The number of GFP-LC3 dots per cell was scored on 50 to 100 cells following a 72 h transfection with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and a 4 h treatment with complete medium (CM), starved medium (EBSS) and starved medium supplemented with 10 mM Leucine (EBSS+Leu). Columns: mean; bars: SEM (n = 3); **p < 0.01. (B) Immunoblot analysis of LC3-I and LC3-II levels in HeLa cells transfected with control (ct) siRNA or GLUD1 siRNA for 72 h and cultured in EBSS supplemented with 10 mM leucine or valine for 4 h. 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 < 0.01. (C) Immunoblot analysis of SQSTM1 levels in HeLa cells transfected with control (ct) siRNA (upper histogram) or GLUD1 siRNA (lower histogram) and cultured for 4 h in complete medium or EBSS supplemented with 10 mM leucine. 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 < 0.01. (D) Measurement of the degradation of long-lived proteins degradation in HeLa cells transfected with control (ct) siRNA (upper western blot and histogram) or GLUD1 siRNA (lower western blot and histogram) for 72 h and cultured in complete medium or EBSS supplemented with 10 mM leucine or 10 mM 3-methyladenine (3-MA). Columns: mean; bars: SEM (n = 3); *p < 0.05.
Mentions: We next investigated whether the regulation of autophagy by leucine is dependent on the expression of GLUD1. For this purpose, we assessed the effect of leucine on autophagy in both control cells and in GLUD1-depleted cells (Fig. 3). Again, in nutrient-starved control cells, the presence of leucine was sufficient to inhibit the biosynthesis of autophagosomes as judged by the number of GFP-LC3 puncta (Fig. 3A). In agreement, leucine normalized the autophagic flux to the level observed in cells cultured in complete medium, as shown by the absence of LC3-II accumulation (Fig. 3B) and impaired degradation of SQSTM1 (Fig. 3C). Finally, leucine also prevented starvation-induced long-lived protein degradation (Fig. 3D). Importantly, we found that knockdown of GLUD1 abolished the inhibitory effect of leucine on the accumulation of GFP-LC3 puncta (Fig. 3A). The increase in autophagosomes correlated with a sustained autophagic flux and lysosomal activity as determined by the accumulation of LC3-II in bafilomycin A1-treated cells (Fig. 3B) and by the degradation of SQSTM1 (Fig. 3C), which could no longer be suppressed by leucine in GLUD1-depleted cells. Similarly, the rate of starvation-induced long-lived protein degradation in GLUD1-depleted cells was shown to be insensitive to leucine addition, but remained sensitive to 3-MA (Fig. 3D), an inhibitor of autophagosome formation by interfering with the activity of PIK3C3 (the class III PtdIns3K).33 Taken together, these results strongly suggest that GLUD1 is required for the inhibitory effect of leucine on 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
Related in: MedlinePlus