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

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.

Figure 1. Role of Sirtuins within Metabolism of Nicotinic Acid

Metabolites are given in black letters, enzymes are given in blue letters.

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. 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. 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. 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. 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.