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. 2013 Nov;9(11):693-700.
doi: 10.1038/nchembio.1352. Epub 2013 Sep 29.

Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide

Kathrin Schmeisser  1 , Johannes Mansfeld, Doreen Kuhlow, Sandra Weimer, Steffen Priebe, Ines Heiland, Marc Birringer, Marco Groth, Alexandra Segref, Yariv Kanfi, Nathan L Price, Sebastian Schmeisser, Stefan Schuster, Andreas F H Pfeiffer, Reinhard Guthke, Matthias Platzer, Thorsten Hoppe, Haim Y Cohen, Kim Zarse, David A Sinclair, Michael Ristow
Affiliations

Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide

Kathrin Schmeisser et al. Nat Chem Biol. 2013 Nov.

Abstract

Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

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Conflict of interest statement

Conflict of interests: D.S. is a consultant and inventor on patents licensed to GlaxoSmithKline, PA, a company developing sirtuin-based medicines.

Figures

Figure 1
Figure 1. Role of Sirtuins within Metabolism of Nicotinic Acid
Metabolites are given in black letters, enzymes are given in blue letters.
Figure 2
Figure 2. Effects of nicotinic acid (NA), nicotinamide (NAM), and 1-methylnicotinamide (MNA) on C. elegans lifespan in the presence and absence of sir-2.1
Lifespan analyses of a wild-type (wt) nematodes exposed to 1 µM MNA (red) compared with untreated worms (black); b of wt nematodes exposed to 100 µM NAM (green); c of wt nematodes exposed to 1 mM NA (blue); d of sir-2.1(ok434) nematodes exposed to 1 mM NA; e of sir-2.1(ok434) nematodes exposed to 100 µM NAM; f of sir-2.1(ok434) nematodes exposed to 1 µM MNA; g of sir-2.1 overexpressing nematodes (strain GA468; orange) compared to rol-6 control worms (black); h of sir-2.1 overexpressing nematodes (strain LG389; orange) compared to control worms (strain LG390; black); i of sir-2.1 overexpressing nematodes (strain GA468) exposed to 1 mM NA; j of control LG390 nematodes exposed to 1 mM NA; k of rol-6 control nematodes exposed to 1 mM NA; l of control LG390 nematodes exposed to 1 mM NA. All data were expressed as mean values with n representing the number of independent experiments. Please find further information to statistical analyses in Suppl. Table 1 (also applies to all following C. elegans lifespan assays).
Figure 3
Figure 3. Disruption and overexpression of nicotinamide-N-methyltransferase/ANMT-1 indicate that 1-methylnicotinamide (MNA) is key regulator of longevity in wild-type and sir2.1-overexpressing nematodes
Lifespan analyses a of anmt-1(gk457) nematodes exposed to 1 mM nicotinic acid (NA; blue); b of anmt-1(gk457) nematodes exposed to 100 µM nicotinamide (NAM; green); c of anmt-1(gk457) nematodes exposed to 1 µM MNA (red). d Expression pattern of GFP as a fused surrogate marker of ANMT-1 protein expression in anmt-1 OE::GFP. Scale bar, 100 µm. e Lifespan analyses of anmt-1 OE::GFP (dark red) compared with wt nematodes (black). f HPLC-derived MNA signals in anmt-1(gk457) nematodes (grey), unsupplemented wt worms (black) and MNA-spiked wt extracts (orange) as well as unsupplemented anmt-1 OE::GFP nematodes (dark red). g Average crawling speed of wildtype (wt) nematodes exposed to NA and MNA expressed as mean values with standard deviation of 3 independent experiments and 10 examined nematodes per condition each. h Lifespan analyses of sir-2.1 overexpressing and anmt-1 deficient nematodes (sir-2.1 OE × anmt-1, strain MIR22; turquoise) compared to rol-6 control worms (black).
Figure 4
Figure 4. 1-Methylnicotinamide (MNA) serves as a substrate for aldehyde oxidase/GAD-3 to form hydrogen peroxide
a ROS levels in wild-type (wt) nematodes following exposure to 1 µM MNA for 4 hrs (red bar) compared with untreated nematodes (black bar). b H2O2 production following exposure to 1 µM MNA for 4 hrs. Data represent mean values with standard deviation of at least 2 independent experiments. c Complex I activity after treatment with 1, 10, and 100 µM MNA; the complex I-inhibitor rotenone (1 µM) served as positive control. Data represent mean values with standard deviation of at least 2 independent experiments and n=4 each. Lifespan analyses d of wt nematodes treated with RNAi against gad-3 exposed to 1 µM MNA; e of sir-2.1 overexpressing nematodes (strain GA468) treated with RNAi against gad-3 (orange); f of wt nematodes exposed to MNA (1 µM) in the presence (purple) or absence of IsoVan (red) compared with worms treated with IsoVan only; g of wt nematodes exposed to the AOX1/GAD-3 substrate vanillin (1 µM, pink). h ROS levels in wild-type (wt) nematodes following exposure to 1 µM vanillin for 24 hrs (pink bar) compared with untreated nematodes (black bar). Data represent mean values with standard deviation of 2 independent experiments.
Figure 5
Figure 5. 1-Methylnicotinamide (MNA) induces a transient ROS signal which is crucial for C. elegans lifespan extension
a ROS levels following 1 mM nicotinic acid (NA; blue bars) exposure and co-treatment with gad-3 RNAi (orange bars) compared with untreated wild-type (wt) nematodes (black bars) at different time points. 1 hr paraquat treatment (PQ, grey bar) acts as positive control. b ROS levels following 1 µM MNA (red bars) exposure at different time points. c ROS levels following 4 hr NA and MNA exposure of anmt-1(gk457) nematodes. d Constitutive ROS levels of wt and anmt-1 OE::GFP nematodes (dark red). e Lifespan analyses of wt nematodes exposed to MNA in the presence (purple) or absence of the antioxidant BHA (red) compared with BHA-treated worms (grey). f Fluorescent microphotograph (enlargement 10-fold) of gst-4::GFP nematodes after 48hr MNA treatment. Scale bar, 100 µm. g Western blot against GFP resembling GST-4 promoter activation in gst-4::GFP nematodes in the presence or absence of skn-1 RNAi after 48 hr MNA treatment compared with untreated nematodes. 48 hr arsenite (As) treatment acts as positive control. h Activity of catalase (CAT) in wt nematodes exposed to 1 µM MNA (red bars). Data represent mean values with standard deviation of at least 2 independent experiments. i Survival of wt nematodes in liquid medium containing 50 mM paraquat after 7 days MNA exposure in comparison with untreated nematodes. j Survival of anmt-1 OE::GFP nematodes in comparison with wt nematodes. Data were expressed as mean values of 2 independent experiments and 50 examined nematodes/condition each.
Figure 6
Figure 6. An acetylation-independent mechanism for sirtuin function in extending lifespan
Turnover of the sirtuin cofactor NAD+ to NA and subsequent irreversible methylation to MNA results in generation of hydrogen peroxide by GAD-3 and a downstream mitohormetic response yielding increased stress resistance.
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