1. Volume 27, Issue 4, Pages 914-925.e5 (April 2018)
Impairing L-Threonine Catabolism Promotes Healthspan through Methylglyoxal- Mediated Proteohormesis 
Meenakshi Ravichandran, Steffen Priebe, Giovanna Grigolon, Leonid Rozanov, Marco Groth, Beate Laube, Reinhard Guthke, Matthias Platzer, Kim Zarse, Michael Ristow 
Cell Metabolism 
Volume 27, Issue 4, Pages e5 (April 2018)
DOI: /j.cmet
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2. Cell Metabolism 2018 27, 914-925.e5DOI: (10.1016/j.cmet.2018.02.004)
Copyright © 2018 Elsevier Inc. Terms and Conditions

3. Figure 1
Impairing gcat/T25B9.1 Expression Extends Lifespan and Promotes Health
(A) Threonine is catabolized to glycine and acetyl-CoA by the enzymes threonine dehydrogenase and glycine-C-acetyltransferase (GCAT) via the unstable intermediate 2-amino 3-ketobutyrate.
(B–F) (B) Effects of gcat/T25B9.1 RNAi (red) versus control (black) on lifespan (p < , log rank test); color coding applies to all subsequent panels and figures. Effect of gcat/T25B9.1 RNAi versus control nematodes regarding (C) aging pigments (∗∗p < 0.005, Student's t test, n = 8 wells × ∼100 worms each) (D), average speed (∗p < 0.01, Student's t test, n = 52), (E) pharyngeal pumping rates (p = 0.51, Student's t test, n = 8 worms × 3 measurements each), and (F) fat content (∗∗p < 0.005, Student's t test, n = 3 worm pellets). Error bars represent the mean ± SD.
(G) Lifespan analysis of gcat/T25B9.1 RNAi (p < 0.0001, log rank test) versus control RNAi in daf-2(e1370) mutant nematodes.
(H) Lifespan analysis of gcat/T25B9.1 RNAi (p < 0.0001, log rank test) versus control RNAi in daf-16(mu86) mutant nematodes.
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4. Figure 2
Methylglyoxal Formation Is Necessary for gcat/T25B9.1 RNAi-Mediated Lifespan Extension
(A) Lifespan analysis of amx-1/amx-2 double mutant treated with control RNAi and amx-3 (black) RNAi versus amx-3 and gcat/T25B9.1 RNAi (red) (p = 0.87, log rank test), respectively.
(B) Relative Amplex Red fluorescence in supernatants of alive nematodes (∗p < 0.05, Student's t test, n = 3 biological replicates from ∼3000 worms each); 100% reflects 1.21 pmol H2O2 in the supernatant per microgram of worm protein. Error bars represent the mean ± SD.
(C) Lifespan analysis of N2 Bristol nematodes treated with gcat/T25B9.1 RNAi the presence (blue) or absence of the antioxidant BHA (red) compared with BHA treated worms (gray).
(D) Lifespan analysis of N2 Bristol nematodes treated with gcat/T25B9.1 RNAi the presence (blue) or absence of the antioxidant NAC (red) compared with NAC treated worms (gray).
(E) HPLC-based measurement of methylglyoxal levels in gcat/T25B9.1 RNAi versus control (∗p < 0.05, Student's t test, n = 5 worm pellets). Error bars represent the mean ± SD.
(F) Lifespan analysis of gcat/T25B9.1 RNAi (blue) versus control RNAi (gray) on a glod-4 overexpressing strain (VH725).
(G) Lifespan analysis of glod-4 RNAis (from the Ahringer [purple] and ORF [orange] libraries) versus control RNAi (black) in N2 Bristol nematodes (both p > 0.05, log rank tests).
(H and I) Lifespan analyses of glod-4 RNAis (from the Ahringer [H] and ORF [I] libraries) combined with gcat/T25B9.1 RNAi (blue) and control RNAi (purple, orange), respectively, in N2 Bristol nematodes (both p < , log rank tests).
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5. Figure 3
MGO Affects Lifespan in a Nonlinear (i.e., Hormetic) Dose-Response Manner
(A and B) Effects of MGO supplementation at increasing concentrations (50 μM–10 mM) on N2 Bristol lifespan, depicted by (A) lifespan curves on heat-inactivated E. coli OP50 (log rank tests) and (B) effects on mean lifespan (∗p < 0.05, ∗∗∗p < by log rank tests). Error bars represent the mean ± SEM.
(C–F) (C) Average speed (∗p < 0.05, Student's t test, n = 107), (D) aging pigments (p = 0.06, Student's t test, n = 7 wells × ∼100 worms each), (E) pharyngeal pumping rates (p = 0.28, Student's t test, n = 8 worms × 3 measurements) and (F) fat content (p = 0.62, Student's t test, n = 3 worm pellets). (C–F) were performed on heat-inactivated E. coli OP50; error bars represent the mean ± SD.
(G) Lifespan analyses of glod-4 RNAi (from the Ahringer library) combined with 100 μM MGO (red) or water (purple), respectively, in N2 Bristol nematodes (log rank test).
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6. Figure 4
Lifespan Extension of gcat/T25B9.1 RNAi and MGO Treatment Is Mediated by the Transcription Factors SKN-1 and HSF-1
(A) Differentially expressed genes as quantified by deep sequencing analysis upon treatment with gcat/T25B9.1 RNAi; black dots indicate no differential regulation, dark blue and light blue dots indicate regulation according to edgeR and DEseq analyses, respectively.
(B) Lifespan analysis of gcat/T25B9.1 RNAi (red) versus control RNAi (black) in skn-1(zu135) mutant nematodes, on alive E. coli HT115 (p = 0.94, log rank test).
(C) Lifespan analysis in skn-1(zu135) mutant nematodes without treatment (black) as well as treatment with 50 μM MGO, on heat-inactivated E. coli OP50 (log rank test).
(D and E) (D) Expression and nuclear localization of GFP in a gst-4-reporter at control conditions and after exposure to 5 days gcat/T25B9.1 or control RNAi (left set of panels), as well as after exposure to 100 μM MGO (right set of panels) (white size bars reflect 200 μm), and (E) corresponding quantifications (∗∗∗p < , χ2 test).
(F) Lifespan analysis of gcat/T25B9.1 RNAi versus control RNAi in hsf-1(sy441) mutant nematodes, on alive E. coli HT115 (p = 0.55, log rank test).
(G) Lifespan analysis in hsf-1(sy441) mutant nematodes without treatment (black) as well as treatment with 50 μM MGO (red), on heat-inactivated E. coli OP50 (p = 0.09, log rank test).
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7. Figure 5
Ubiquitin-Proteasome Pathway Is Required for gcat/T25B9.1-Mediated Effects on Healthspan
(A–G) (A) qPCR analysis of proteasome subunits upon after exposure to 5 days gcat/T25B9.1 RNAi versus control (∗p < 0.05, xp = 0.10, Student's t test, n = 3 biological replicates each). Error bars represent the mean ± SD. Lifespan analysis in N2 Bristol nematodes the co-presence of gcat/T25B9.1 RNAi versus control RNAi with (B) pbs-3 RNAi (p = 0.35, log rank test), (C) rpn-6.1 RNAi (p = 0.42, log rank test), and (D) pbs-4 RNAi (p = 0.058, log rank test). Lifespan analysis in N2 Bristol nematodes exposed to 100 μM MGO together with (E) pbs-3 RNAi (p = 0.086, log rank test), (F) rpn-6.1 RNAi (p = 0.26, log rank test), and (G) pbs-4 RNAi (p = 0.575, log rank test), or to respective control RNAi, on alive E. coli HT115.
(H–J) (H) Chymotrypsin-like, (I) caspase-like, and (J) trypsin-like activities of proteasome in N2 Bristol nematodes treated with gcat/T25B9.1 RNAi versus control (∗p < 0.05, Student's t test, n = 3 worm pellets each). Error bars represent the mean ± SD.
(K–M) (K) Chymotrypsin-like (∗p < 0.05), (L) caspase-like (p = 0.63) and (M) trypsin-like (p = 0.09) activities of proteasome in N2 Bristol nematodes treated with 50 μM MGO (for all: Student's t test, n ≥ 4 worm pellets each). Error bars represent the mean ± SD.
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8. Figure 6
Mechanistic Summary for gcat/T25B9.1- and MGO-Mediated Longevity
Impaired expression of gcat/T25B9.1 leads to increased formation of hydrogen peroxide and methylglyoxal in an AMX-dependent manner. Subsequently, and dependent on MGO-induced transcription factors, namely SKN-1 and HSF-1, activation of the ubiquitin-proteasome system mediates extension of lifespan and associated health parameters in nematodes.
Cell Metabolism  , e5DOI: ( /j.cmet )
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