compartments for different functions ( 4, 5). Of
the nine C. elegans FABP homologs, only mRNA
amounts of lbp- 8 were increased in lipl- 4 Tg animals, but not in the lipl- 4 Tg no SP strain (Fig. 1F).
A green fluorescent protein (GFP) reporter strain
showed that lbp- 8 was exclusively expressed in
the intestine (fig. S5A). Both FLAG- and mCherry-tagged LBP- 8 proteins were predominantly localized to intestinal lysosomes (Fig. 1, G to I, and
fig. S5, B to J).
We also detected partial nuclear localization of
LBP- 8 in the intestine, which was enhanced in
lipl- 4 Tg animals (Fig. 2, A to G, and fig. S6, A to
F). LBP- 8 contains an N-terminal nuclear localization signal (NLS) (fig. S6G) and was present in
both cytoplasmic and nuclear fractions of total
worm lysate (fig. S6H). Both RNA interference
(RNAi)–mediated depletion of LBP- 8 and a newly isolated deletion mutant, lbp- 8(rax1), suppressed
the life-span extension in lipl- 4 Tg animals without
affecting WT life span (Fig. 2H, fig. S7, and table
S1). Thus, LBP- 8 appears to be required for LIPL- 4
lysosomal activity to confer longevity.
We found that a transgenic strain (lbp- 8 Tg)
constitutively expressing lbp- 8 had a 30% increase
in mean life span compared with WT animals
(Fig. 2I and table S1) and improved maintenance
of physical activity in old age (fig. S3B). However,
a transgenic strain that constitutively expresses
LBP- 8 lacking NLS (lbp- 8 Tg no NLS) was excluded from nuclei and showed little or no life-span extension (fig. S8 and table S1). Thus, LBP- 8
may function as a lysosomal lipid chaperone
transducing lipid signals to the nucleus.
To test whether lysosomal signals might influence nuclear transcription, we screened several transcription factors implicated in longevity
regulation ( 6–11). Nuclear hormone receptors
nhr- 49 and nhr- 80, previously demonstrated to
physically interact ( 10), were both required for
lipl- 4– and lbp- 8–mediated longevity. RNAi-mediated inactivation of nhr- 49 in adult worms
shortened the life span of WT worms but also
completely suppressed longevity extension in
lipl- 4 Tg and lbp- 8 Tg worms (Fig. 3A, fig. S9A
and table S1). The loss-of-function mutation
nhr- 80(tm1011) abrogated longevity extension
without affecting the life span of WT worms
(Fig. 3B, fig. S9B, and table S1). Neither nhr- 49
nor nhr- 80 is required for dietary restriction–
induced longevity ( 6, 12), which suggests that
the LIPL- 4–mediated longevity mechanism may
act independently of dietary restriction. Concordantly, the longevity extensions by lipl- 4 Tg and
eat-2(ad1116), a genetic model of dietary restriction in C. elegans ( 13), were additive (fig. S10).
acs-2 encodes an acyl-CoA synthetase required
for mitochondrial b-oxidation and is a target
gene of NHR- 49 ( 11). acs-2 transcription was
increased more than 15-fold in lipl- 4 Tg animals;
this effect was dependent on nhr- 49 and nhr- 80,
and absent in the lipl- 4 Tg no SP strain (Fig. 3C).
Transcription of acs-2 was also increased more
than 10-fold in lbp- 8 Tg but not in lbp- 8 Tg no
NLS animals (Fig. 3D). Thus, LIPL- 4–induced
activation of NHR- 49 and NHR- 80 can be re-
produced by nuclear action of LBP- 8. Transcrip-
tional increase of lbp- 8 by lipl- 4 Tg was in turn
mediated by NHR- 49 and NHR- 80 (Fig. 3E).
To identify lipid molecules that might func-
tion in this lysosome-to-nucleus lipid signaling,
we performed high-throughput metabolomic
profiling analyses on WT and lipl- 4 Tg worms.
Among 352 metabolites detected, 71 had sig-
nificantly altered abundance in lipl- 4 Tg ani-
mals (table S2). Long-chain fatty acids and their
derivatives are likely binding partners of FABPs
( 4). Thus, we focused our analysis on three C20
fatty acids—arachidonic acid, w- 3 arachidonic acid,
and dihomo-g-linolenic acid—and oleoylethanol-
amide (OEA), an N-acylethanolamine fatty acid
derivative (Fig. 4A and table S2). In fluorescence-
based binding assays, all four lipids bound to
LBP- 8, and the binding affinity of OEA for LBP- 8
was 3 times that of the fatty acids (Fig. 4B).
Next, we tested the effects of the four lipids on
transcription when directly applied to WT adult
worms. We also used an OEA analog, KDS-5104,
that is more resistant to hydrolysis than OEA
( 14). Only OEA and its analog were sufficient to
increase the transcription of lbp- 8 in W T worms,
and the analog exerted a stronger effect (Fig. 4C).
After 3 hours of treatment with the OEA analog,
transcription of lbp- 8 and acs-2 was increased
more than 4- and 7-fold above the control levels,
respectively (Fig. 4, D and E). This effect was
abrogated in the nhr- 49(nr2041) or nhr- 80(tm1011)
mutant (Fig. 4, D and E). Thus, accumulation of
OEA in response to LIPL- 4 may act to promote
transcription via NHR-49/NHR- 80.
To test whether OEA directly binds to NHR- 49
or NHR- 80, or both, we measured intrinsic fluorescence changes of glutathione S-transferase
(GST)–NHR fusion proteins in the presence of
OEA. OEA binding significantly decreased the
fluorescence intensity of the NHR- 80 fusion protein in a dose-dependent manner [equilibrium
dissociation constant (Kd) of 7. 8 mM] (Fig. 4F).
In a differential protease-sensitivity assay, chymotrypsin digestion of [35S]NHR- 80 in the presence
of the OEA analog resulted in protease-resistant
fragments of approximately 45 and 35 kD (fig.
S11), which indicated direct binding between
NHR- 80 and the OEA analog. However, no binding was detected between NHR- 49 and OEA or
the OEA analog (fig. S12). Thus, NHR- 80 appears
to act as a direct nuclear receptor of OEA and
NHR- 49 may function as a cofactor of NHR- 80.
N-Acylphosphatidylethanolamine–specific phos-pholipase D (NAPE-PLD) mediates OEA synthesis
( 15). In C. elegans, nape-1 and nape-2 encode NAPE-PLD ( 16). The nape-1(tm3860) loss-of-function
mutation suppressed the life-span extension in
lipl- 4 Tg and lbp- 8 Tg by half (fig. S13 and table
S1). Additionally, a loss-of-function mutant lipl- 4
(tm4417) reduced the longevity of lbp- 8 Tg by 68%
(fig. S14), which supports the possibility that LIPL- 4
activity promotes the generation of longevity-promoting OEA carried by LBP- 8.
Direct treatment of WT worms with the OEA
analog prolonged life span (Fig. 4G and table S1)
and improved physical activity maintenance in
aged animals (Fig. 4H). In contrast, neither lipl- 4
Tg nor lbp- 8 Tg life span was affected by OEA
Overall, our studies suggest that bioactive lipid
messengers and lipid chaperones link lysosomal
activity and nuclear transcription to promote
longevity. All the components of this lysosome-
to-nucleus signaling pathway are well conserved
in mammals. Notably, mammalian peroxisome
proliferator–activated receptor a is activated by
OEA ( 17), whereas NHR- 80 is homologous to mam-
malian HNF4, which suggests that different nu-
clear receptors bind the same ligands despite
divergent ligand-binding domains. Considering
that FABPs are quite promiscuous in ligand bind-
ing ( 4), there may be other lipid molecules bind-
ing to LBP- 8 and functioning in this longevity
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We thank H. Y. Mak and A. Antebi for providing strains; A. Dervisefendic
and H. Jen for experimental support; N. Timchenko, J. Wang, Z. Yu,
and D. Chow for instrumental support; H. Dierick, C. Herman,
H. Zheng, F. Xia, S. Rosenberg, and H. Zoghbi for critical reading
of the manuscript; P. P. Metoyer for scientific editing. Supported by
NIH grants T32GM008602 (E.H.A.), R01DK095750 (E.A.O.),
T32HD055200 (A.F.), F30AG046043 (A.F.), R00AG034988
(M.C. W.), and R01AG045183 (M.C. W.); Ellison New Scholar Award
(M.C. W.); Welch Chair in Science (Q-0022) (D.D.M.); European
Research Council Advanced Grant (R.Z.); and Fondation Leducq
(R.Z.). Requests for materials should be addressed to M.C. W.
Materials and Methods
Figs. S1 to S16
Tables S1 and S2
References ( 19–23)
16 July 2014; accepted 13 November 2014
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