D-beta-hydroxybutyrate extends lifespan in C. elegans

Abstract

The ketone body beta-hydroxybutyrate (βHB) is a histone deacetylase (HDAC) inhibitor and has been shown to be protective in many disease models, but its effects on aging are not well studied. Therefore we determined the effect of βHB supplementation on the lifespan ofC. elegans nematodes. βHB supplementation extended mean lifespan by approximately 20%. RNAi knockdown of HDACs hda-2 or hda-3 also increased lifespan and further prevented βHB-mediated lifespan extension. βHB-mediated lifespan extension required the DAF-16/FOXO and SKN-1/Nrf longevity pathways, the sirtuin SIR-2.1, and the AMP kinase subunit AAK-2. βHB did not extend lifespan in a genetic model of dietary restriction indicating that βHB is likely functioning through a similar mechanism. βHB addition also upregulated ΒHB dehydrogenase activity and increased oxygen consumption in the worms. RNAi knockdown of F55E10.6, a short chain dehydrogenase and SKN-1 target gene, prevented the increased lifespan and βHB dehydrogenase activity induced by βHB addition, suggesting that F55E10.6 functions as an inducible βHB dehydrogenase. Furthermore, βHB supplementation increased worm thermotolerance and partially prevented glucose toxicity. It also delayed Alzheimer's amyloid-beta toxicity and decreased Parkinson's alpha-synuclein aggregation. The results indicate that D-βHB extends lifespan through inhibiting HDACs and through the activation of conserved stress response pathways.

Figure 1

D-βHB extends the lifespan of N2 C. elegans worms. (A) Concentration dependency of βHB-mediated extension of lifespan. (B) D-βHB, but not L-βHB addition led to lifespan extension. When no D or L prefix is present, βHB refers to DL-βHB.

Figure 2

βHB-mediated HDAC inhibition plays a role in lifespan extension. (A) Survival of N2 worms in the presence of βHB, butyrate, or both compounds together. (B) Effects of hda-1, (C) hda-2, or (D) hda-3 RNAi knockdown on C. elegans lifespan in the presence or absence of 20 mM βHB.

Figure 3

D-βHB dehydrogenase activity in worm extracts following RNAi-mediated gene knockdown. (A) D-βHB dehydrogenase activity following worm culture in the absence or presence of 10 mM D-βHB. Conditions in the legend refer to the culture conditions. The genes F55E10.6, dhs-2, dhs-16, or dhs-20 were knocked down by RNAi feeding (* p < 0.05 compared to Control + 10 mM D-βHB; # p < 0.05 compared to Control). (B) D-βHB dehydrogenase activity following worm culture in the absence or presence of 10 mM L-βHB. The assay conditions were the same as panel A (# p < 0.05 compared to Control).

Figure 4

F55E10.6 is required for βHB-mediated lifespan extension, but not for βHB-induced oxygen consumption. (A) Treatment with βHB did not increase the lifespan of N2 worms fed RNAi to F55E10.6. (B) The addition of N-acetyl-L-cysteine (NAC) did not decrease the lifespan of βHB treated worms. (C) The effect of 20 mM βHB and RNAi knockdown of F55E10.6 on oxygen consumption (* p < 0.05 vs. untreated; # p < 0.05 vs. Control). (D) The effect of 20 mM βHB treatment on ATP levels in day 4 N2 worms (p = 0.202). Data are represented as mean +/− SEM.

Figure 5

SKN-1 and DAF-16 are required for βHB-mediated lifespan extension. (A) βHB addition did not increase the lifespan of N2 worms fed RNAi to knockdown expression of skn-1. (B) βHB addition increased fluorescence of the gst-4::gfp SKN-1 reporter strain. Data are represented as mean +/− SEM (* p < 0.05). (C) βHB addition did not increase lifespan in daf-16(mgDf50) mutant worms. (D) βHB or butyrate increased fluorescence when administered to the sod-3::gfp DAF-16 reporter strain. Data are represented as mean +/− SEM (* p < 0.05).

Figure 6

Treatment with βHB increases thermotolerance (log-rank p < 0.001) in N2 worms when upshifted from 20°C to 35°C. βHB mean survival time = 5.7 hours. Control mean survival time = 4.5 hours.

Figure 7

A decreased rate of protein synthesis contributes to βHB-mediated longevity. (A) βHB-mediated lifespan extension was blunted in rsks-1(ok1255) mutant worms. (B) βHB-mediated lifespan extension was also blunted in gcn-2(ok871) mutant worms.

Figure 8

βHB extends the lifespan of short-lived mitochondrial ETC complex I and complex II mutants. (A) gas-1(fc21) survival in the absence and presence of βHB. (B) mev-1(kn1) survival in the absence and presence of βHB.

Figure 9

βHB extends lifespan in a similar manner as DR and requires AAK-2, SIR-2.1, and CBP-1. (A) βHB does not extend lifespan of AMPK mutant aak-2(TG38) worms, (B) sir-2.1(ok434) worms, or (C) eat-2(ad1116) worms. (D) Additionally, βHB does not extend the lifespan cbp-1 RNAi knockdown N2 worms.

Figure 10

βHB protects against glucose toxicity and proteotoxicity. (A) Treatment with βHB partially protects against 50 mM glucose-induced reduction of lifespan in N2 worms. (B) Survival of the CL4176 strain of C. elegans expressing Aβ in muscle following temperature upshift. Treatment with βHB increases the time to paralysis (log-rank p < 0.001). βHB-treated mean lifespan = 29 hours, untreated control mean lifespan = 26 hours. The curves are generated from the results of six assays (n > 500 for both groups). (C) Treatment with βHB decreases α-synuclein-GFP aggregation in the NL5901 strain GFP fluorescence readings were taken on day 8 of worm lifespan. Data are represented as mean +/− SEM. (log-rank p < 0.001) (D) 20 mM βHB did not protect against the shortened lifespan induced by human TDP-43 overexpression when the worms were grown at 16°C.