(12) or SudanBlack, a fat-specific dye (fig. S2) (13). At the permissive temperature, the glp mutants reproduced normally and their fat storage was similar to that of the wild type (fig. S3).Fat storage can be altered by changes in either energy input or expenditure. Food intake and retention in the gut are unchanged in glp-1 (Fig. 1, H and J); the food absorption rate is also normal (Fig. 1I). Normal locomotion in glp-1 suggests that less fat storage is not due to an increase in physical activity (Fig. 1K). Therefore, decreased fat storage in the germ line–defective mutants is unlikely to be the result of alterations in energy intake and/or physical activity, and more likely reveals an altered endocrine signaling axis.Production of vitellogenin-rich oocytes is the most energy-intensive reproductive function. We used fem-3 sterile mutants to examine whether gametogenesis influences fat storage. The gain-of-function allele fem-3(q20ts) produces only sperm, whereas the loss-of-function allele fem-3(e2006ts) produces only oocytes at the nonpermissive temperature (14,15). Neither fem-3mutant exhibited abnormal lipid accumulation (Fig. 2, A to D). This result excludes the possibility that gametogenesis regulates fat storage, and it also suggests that sterility per se does not cause a change in lipid accumulation.To test whether germline proliferation regulates fat storage, we shifted glp-1 mutants to the restrictive temperature at different evelopmental stages to arrest germline proliferation at distinct points. Adults that are generated from L2 (early) temperature shifts carry few mitotic germ cells, whereas adults from L4 (late) temperature shifts form the germ line with essentiall
y wild type– sized mitotic and meiotic germ cells and differentiated sperm. Despite a very different composition of the germ line, adult fat storage was decreased to a similar extent under all conditions (Fig. 2E). One process shared by all temperature shifts is germline stem cell (GSC) arrest (16), which could induce the decrease in fat storage. We therefore shifted temperature at 1 day of adulthood, after animals started to reproduce; this should affect adult GSCs but not the already proliferated germ line. By 30 hours at the restrictive temperature, fat storage in glp-1 started to decrease (Fig. 2F). Within 48 hours, lipid accumulation in glp-1 was
reduced to an extent comparable to that seen with the developmental temperature shifts (Fig.
2F). This result suggests that GSCs regulate fat storage during adulthood.
The somatic distal tip cell forms the niche of GSCs. The Notch ligand LAG-2 expressed in the
distal tip cell is required to maintain GSC identity (17). Like glp-1 mutants, lag-2(q420ts)
mutants (18) showed a 50% decrease in fat storage (Fig. 2G). glp-1(ar202gf) mutants with a
hyperactive GLP-1, in which entry into meiosis is prevented and GSCs overproliferate (19),
showed a factor of 1.7 fat increase (Fig. 2, H, I, and K), which suggests that a deficit of GSCs
signals low fat storage and that GSC overproliferation signals high fat storage. No change in
fat content was detected in gld-1(q485) mutants, in which early-phase meiotic germ cells
reenter into the mitotic cell cycle and overproliferate (20) (Fig. 2, H, J, and K). Thus, once
germ cells undergo differentiation, they lose the ability to modulate fat storage.
To understand the mechanisms by which GSCs regulate fat storage, we reduced the activities
of 163 metabolic genes by RNA interference (RNAi) and screened for gene inactivations that
increase fat storage in glp-1 (table S1). Among 16 potential candidate genes identified,
K04A8.5 encoded a triglyceride lipase, which most strongly affected fat storage. Inactivation
of K04A8.5 partially restored fat storage in glp-1 but had marginal effect on the wild type (Fig.
3A). GSC arrest caused a marked increase in the transcriptional levels of K04A8.5 (Fig. 3B),
and a promoter–green fluorescent protein (GFP) reporter that was not detected under normal
conditions became detectable in the glp-1 gut at the restrictive temperature (fig. S4). High gene
dosage of K04A8.5 decreased fat storage in the wild type, and genetic mosaic animals showed
that intestinal cells that constitutively express K04A8.5 had fewer lipid droplets than did
neighboring nontransgenic cells (Fig. 3, C to E). These results imply that this lipase acts in fat
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storage tissue rather than in endocrine cells or GSCs. Thus, the decrease in fat storage upon
GSC arrest is induced by increased lipid hydrolysis via up-regulation of K04A8.5.
GSC arrest caused by glp-1 loss of function resulted in extended life span (Fig. 3F and table S2) (8); K04A8.5 RNAi suppressed this increased longevity but did not reduce wild-type life span (Fig. 3F and table S2). Therefore, up-regulation of this lipase gene mediates both lipid hydrolysis and longevit
y in GSC-arrested animals. Constitutive expression of K04A8.5specifically in the intestine led to life spans that were 24% longer than in control siblings (Fig.3G and table S3). Thus, lipid hydrolysis in fat storage tissue prolongs life span, which connects the metabolic functions of adipose tissue to life-span control.We investigated the signaling pathways regulating K04A8.5 expression in the intestine. The forkhead transcription factor DAF-16 is translocated into nuclei in the intestine upon GSC arrest (21). To test whether daf-16 is involved in regulation of fat storage by GSC proliferation,we inactivated daf-16 by RNAi in wild-type and glp-1 mutants and assayed fat storage.daf-16 inactivation restored fat storage in glp-1 but did not affect wild-type fat storage (Fig.4A and fig. S5). K04A8.5 up-regulation in glp-1 was abolished in the absence of daf-16 but was not altered in wild-type animals subjected to daf-16 RNAi (Fig. 4B). Thus, upon GSC arrest, DAF-16 is activated in the intestine to promote lipid hydrolysis through induction of K04A8.5 expression. External stresses such as heat shock and oxidative stress activate daf-16 (22,23). After heat shock and paraquat treatment, the DAF-16 targets hsp-16.1 and ctl-2 were up-regulated but K04A8.5 was not (fig. S6). These results suggest a specific regulation of K04A8.5 by the signal from the germ line.KRI-1, the human KRIT 1 homolog, and DAF-12, the nuclear hormone receptor, are both required for the intestinal nuclear localization of DAF-16 in GSC-arrested animals (21). These factors could act upstream of DAF-16 to sense signals from GSC and, in response, regulate lipid accumulation. Like daf-16 RNAi,
kri-1 RNAi significantly reduced K04A8.5 expression and increased the fat content in glp-1 (Fig. 4, A and B, and fig. S5). In contrast, reducing
daf-12 function did not affect K04A8.5 levels and caused a slight decrease in lipid accumulation
in both wild-type and glp-1 mutants (fig. S7). Therefore, GSC arrest promotes lipid hydrolysis
in the intestine through activation of the kri-1/daf-16 signaling pathway, but independently of
daf-12 lipophilic hormone signaling.
We examined K04A8.5 expression in other long-lived animals, such as worms with reducing
function in insulin receptor/daf-2. daf-2 is crucial in regulation of fat metabolism during larval
development (24). Therefore, we reduced daf-2 function only at adulthood by RNAi feeding.
Reducing daf-2 activity at adulthood caused up-regulation of K04A8.5 and decreased fat
storage (Fig. 4, C and D, and fig. S8). Loss of thegerm line and reduced daf-2 signaling
synergistically induced K04A8.5 and decreased fat storage (Fig. 4, C and D, and fig. S8). We
also found that K04A8.5 RNAi partially suppressed the longevity of daf-2 mutants (Fig. 4E
and table S4). These results suggest that lipid hydrolysis is also connected to life-span control
in the daf-2 long-lived animals.
Our findings reveal an endocrine signaling axis from GSCs to fat storage tissue, with feedback
from the fat storage to the longevity of the animal. Somatic stem cells are thought to mediate
tissue regeneration after wounding, and such regeneration is also known to decline with aging.
How the proliferation of adult stem cells is coupled to the requirement for replacement cells
during normal and pathological aging may be related to the metabolic pathways we have
modulatediscovered between germline stem cells and the longevity of C. elegans.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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25. We thank H. Mak, J. Dittman, and J. Avruch for critical reading of the manuscript; N. Ringstad for
laser ablation techniques; V. Rottiers, A. Antebi, and the Caenorhabditis Genetics Center for
providing strains; A. Fire for GFP vectors; and members of the Ruvkun lab for discussions. Supported
by Life Sciences Research Foundation and Ellison Medical Foundation fellowships (M.C.W.), a
Human Frontier Science Program fellowship (E.J.O.), and NIH grants 5R01AG016636 and
5R37AG14161 (G.R.).
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Fig. 1. Influence of reproductive activity on fat metabolism (A  to C ) Ablation of germline precursor cells resulted in a 50% reduction in adult fat storage (P  < 0.0001) (n  = 15). (D  to G ) The same degree of reduction was observed in glp-4(bn2) and glp-1(e2141) mutants defective in germline proliferation (P  < 0.0001) (N2 and glp-4, n  = 15;glp-1, n  = 18). (H  to J ) Decreased fat in glp-1 was not due to less food intake (measured as pharyngeal pumping and food absorption rates) or food ret
ention time (measured as defecation rate) (P  > 0.5 for each) (pumping rate, N2, n  = 12; glp-1, n  = 18; food absorption rate, N2 and glp-1, n  = 5; defecation rate, N2 and glp-1, n  = 7). (K ) Comparison of locomotory behavior showed that physical activity does not change in glp-1. Experiments were performed at the restrictive temperature (25°C) for both wild-type (N2) and glp  mutants (n  = 21 for each).NIH-PA Author Manuscript
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