Water sensor ppk28 modulates Drosophila lifespan andphysiology through AKH signalingMichael J. Watersona, Brian Y. Chungb, Zachary M. Harvanekb,c, Ivan Ostojicd, Joy Alcedod,e, and Scott D. Pletchera,b,f,1

aProgram in Cellular and Molecular Biology, bDepartment of Molecular and Integrative Physiology, cMedical Scientist Training Program, and fGeriatricsCenter, University of Michigan, Ann Arbor, MI 48109; dGrowth Control Unit, Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland;and eDepartment of Biological Sciences, Wayne State University, Detroit, MI 48202

Edited* by Cynthia Kenyon, University of California, San Francisco, CA, and approved April 24, 2014 (received for review August 14, 2013)

Sensory perception modulates lifespan across taxa, presumablydue to alterations in physiological homeostasis after central nervoussystem integration. The coordinating circuitry of this control, how-ever, remains unknown. Here, we used the Drosophila melanogastergustatory system to dissect one component of sensory regulationof aging. We found that loss of the critical water sensor, pickpocket28 (ppk28), altered metabolic homeostasis to promote internal lipidand water stores and extended healthy lifespan. Additionally, lossof ppk28 increased neuronal glucagon-like adipokinetic hormone(AKH) signaling, and the AKH receptor was necessary for ppk28 mu-tant effects. Furthermore, activation of AKH-producing cells alonewas sufficient to enhance longevity, suggesting that a perceivedlack of water availability triggers a metabolic shift that promotesthe production of metabolic water and increases lifespan via AKHsignaling. This work provides an example of how discrete gusta-tory signals recruit nutrient-dependent endocrine systems to coor-dinate metabolic homeostasis, thereby influencing long-term healthand aging.

taste | adipokinetic hormone signaling

Sensory signaling systems are potent modulators of organismalmetabolism and lifespan (1–6) but the mechanisms by which

sensory inputs are transduced into relevant physiological outputsremain poorly understood. For even the simplest organisms, anextensive array of sensory stimuli—including chemical, mechani-cal, thermal, and visual cues—must be properly transduced andintegrated to ensure a reliable response to environmental quality.In the nematode Caenorhabditis elegans, sensory neurons alonemay accomplish these tasks. They express multiple sensory recep-tors, which provide simple integrative capabilities to the cell, andthey secrete neuropeptides, which can direct cell-nonautonomousresponses in peripheral tissues (7). The fruit fly, Drosophilamelanogaster, however, is similar to mammals in that sensoryneurons are often highly specialized, and elaborate mechanismsof sensory integration and interpretation are performed by spe-cialized processing centers in the central brain (8). Once deco-ded, sensory signals are presumably relayed to neuroendocrinecenters to stimulate appropriate actions in peripheral tissues.Whereas the release of endocrine molecules, including insulin-like peptides, via central nervous system (CNS) control has emergedas a critical regulator of aging across model organisms, the extentto which sensory signals impact such systems, and the underlyingneurocircuitry involved, are unknown (9–11).The Drosophila system is a powerful tool for elucidating evo-

lutionarily conserved aspects of neural circuitry that link sensoryinformation to a variety of behavioral and metabolic responses.Although comprised of only ∼100,000 neurons, the fly brain issufficiently complex to share many aspects of structure andfunction with humans and mice. This, along with the ability tomanipulate neuronal activity in a temporally and spatially con-trolled manner, has made it an effective tool for elucidatingneural mechanisms of complex behaviors that include, amongothers, feeding (12, 13) and mating (14). We thus inferred that

this system would have high utility for mapping the circuitryunderlying changes in aging due to sensory input.To identify and dissect sensory networks that regulate aging in

the fly, we focused on taste perception. We reasoned that gus-tatory chemosensory signals would be particularly relevant giventheir role in assessing dietary landscape, the composition ofwhich has been found to have profound effects on organismallifespan (15, 16). Additionally, the similarity between taste pro-cessing in Drosophila and mammalian systems suggested that ourwork could be applied broadly. In mammals, for example, tastereceptors (G protein-coupled receptors or ion channels) are seg-regated by modality in specialized epithelial taste receptor cells,and their activation leads to transduction of taste cue to the pri-mary gustatory cortex of the CNS (8). Comparably,D. melanogasteralso maintains specialized and segregated taste cells, expressingeither G protein-coupled gustatory receptors (GRs) or membersof the pickpocket (ppk) family of ion channels (17–19). Thesebona fide neurons directly map to the gustatory center of theDrosophila CNS, the subesophageal ganglion (8). Little is knownin either system, however, about links between the taste pro-cessing centers and well-characterized neuroendocrine cells.As a vital nutrient for all animals, we predicted that water

would be an environmental component capable of driving gus-tatory modulation of physiology and lifespan. The regulation ofwater intake, for example, is essential for organisms to maintainproper osmotic homeostasis, and whole organ systems are de-voted to maintaining its balance. It has long been known thatinsects harbor specialized sensory neurons that are sensitive towater (20, 21). These are concentrated on the proboscis, themajor organ of ingestion of the fly. The proboscis is covered with

Significance

Sensory inputs are known to control aging. The underlyingcircuitry through which these cues are integrated into regula-tory physiological outputs, however, remains largely unknown.Here, we use the taste sensory system of the fruit fly Drosophilamelanogaster to detail one such circuit. Specifically, we findthat water-sensing taste signals alter nutrient homeostasis andregulate a glucagon-like signaling pathway to govern productionof internal water production. This metabolic alteration likelyserves as a response to water sensory information. This controlof metabolic state, in turn, determines the organism’s long-termhealth and lifespan. Our studies, then, provide a framework forunderstanding sensory control of aging as well as several targetsto potentially maximize organismal health.

Author contributions: M.J.W., B.Y.C., J.A., and S.D.P. designed research; M.J.W., B.Y.C.,Z.M.H., and I.O. performed research; M.J.W., B.Y.C., and S.D.P. analyzed data; and M.J.W.and S.D.P. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: spletch@umich.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1315461111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1315461111 PNAS | June 3, 2014 | vol. 111 | no. 22 | 8137–8142

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taste sensilla, with each sensillum harboring up to four GRneurons that each recognize a distinct taste modality (sweet,bitter, pheromone, or water) (17–19). The osmosensitive ionchannel ppk28 is expressed in water-sensitive neurons, and it isrequired for a functional gustatory response to external water(18). Osmosensitive taste receptors have also been identified inthe rat (22, 23) and the ability of water to stimulate gustatorynerve fibers, a response that has been termed “water response,”is common in many vertebrates (22). Finally, water availabilityhas been implicated in Drosophila aging (24, 25).We therefore asked whether sensory perception of water mod-

ulates aging in the fly. We first determined the metabolic andlongevity consequences of loss of function of the gustatory geneppk28. We found that loss of this critical water sensor promotedlongevity, which was accompanied by increased glucose and lipidstores and by enhanced physical performance throughout life.The receptor for the glucagon-like molecule adipokinetic hormone(AKHR) was required for ppk28-mediated effects, as was the in-tegral insulin-like signaling transcription factor, Drosophila ho-molog of FoxO transcription factor (dFOXO). Consistent withthe genetic data, ppk28 mutant flies exhibited an up-regulationof AKH signaling, which is known to promote lipid mobilization(26) and therefore may serve to increase metabolic water pro-duction (27). Indeed, mutant animals were desiccation resistantand retained greater amounts of internal water. Finally, acti-vation of AKH-producing neurons was sufficient to increaselifespan, suggesting that the promotion of longevity in our sen-sory mutants may result from an alteration of physiologic priori-ties to emphasize metabolic water production in response to aperceived environmental scarcity. This work, then, provides aframework for understanding how the perception of nutrients,distinct from consumption, is processed in the CNS to directlongevity-regulating physiological consequences. Furthermore,these data reveal the role that water specifically maintains as aphysiologically influential dietary component.

ResultsLoss of ppk28 Function Increases Drosophila Health and Lifespan. Todetermine whether water-sensing inputs, requiring ppk28 func-tion, were capable of affecting longevity, we acquired a ppk28-null mutant line (ppk28Δ) containing a 1.769-kb deletion sur-rounding the ppk28 endogenous locus (18). Flies lacking ppk28fail to exhibit a functional gustatory response to a water stimulus(18). Deletion mutants were backcrossed to two separate controllines [w1118-VDRC (w) and yw] for six to eight generations withlifespan measured and compared with background controls (Fig.1 A and B). Indeed, female ppk28-null mutant flies showed asignificant increase in mean and maximum lifespan in both geneticbackgrounds (a maximum of 43.55% increase in mean lifespan inw and 24.78% in yw; see Table S1 for replicate experiments). Lossof ppk28 also extended male lifespan in both genetic backgrounds,although to a lesser degree (Fig. S1 A and B). Additionally, asecond ppk28 mutant allele created via P-element insertion intothe third of four exons of the ppk28 gene (ppk28G981) (28) ex-tended female lifespan (a mean lifespan increase of 13.1%)compared with background control (Fig. S1C). To test whetherloss of water-sensing gustatory information was also associatedwith an increase in overall health, we performed a negative geo-taxis assay and found that ppk28 mutants showed greater perfor-mance than background controls throughout their lifespan (Fig.1C). Furthermore, ppk28mutants showed increased resistance tostarvation stress (Fig. 1D).Gustatory manipulations have the potential to alter food in-

take, and dietary restriction is sufficient to affect lifespan acrossmodel organisms (15). Therefore, to determine whether ppk28-null mutants were long-lived simply because they were eating lessthan their background controls, we quantified feeding behaviorin these flies. Our longevity assays used a sugar–yeast medium

containing 10% (wt/vol) of both macronutrients (called “SY10%”),and food intake rates in ppk28 mutant flies were statisticallyindistinguishable from control animals under these conditionsat a young age and significantly increased at an advanced age(Fig. S2A). Furthermore, we found that overall nutrient con-centration (ranging from SY5% to SY15%), and therefore theosmolarity of the medium, had no effect on this relationship(Fig. S2B). These data suggest that ppk28 mutant flies are notlong-lived due to decreased feeding behavior.Improvements in lifespan and health measures in our flies

were attributable to disruption of the ppk28 locus. Transgenicaddition of a small duplication element (Dp) from the firstchromosome inserted onto the third chromosome (1;3) con-taining the endogenous ppk28 locus [Dp(1;3)DC320, “Dp(320)”for brevity] rescued the lifespan extension associated with ppk28deletion. Whereas transgenic stocks were marginally longer livedthan the w1118 stock in general, loss of ppk28 had no effect onlifespan in flies that simultaneously carried a duplication of theregion (Fig. 1 E and F). The presence of the duplication alsoreversed physiological changes associated with ppk28 loss of func-tion (see ppk28-Mediated Signals Regulate Nutrient Homeostasis).Together, these data suggest that integration of ppk28-mediated

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Fig. 1. Loss of ppk28 function increases healthy lifespan in D.melanogaster.(A and B) Kaplan–Meier survival curves for female ppk28 deletion mutants inboth w1118-VDRC (w) [n = 246 (w); n = 248 (w,ppk28Δ); mean lifespan increaseof 20.68 d (43.55%)] (A) and yw [n = 244 (yw); n = 248 (yw,ppk28Δ); meanlifespan increase of 13.48 d (24.78%)] (B) background and their corre-sponding background controls. (C) Analysis of vertical distance climbed inthe longitudinal negative geotaxis assay of ppk28 mutant (w,ppk28Δ) andbackground control (w) female flies (n = 10 groups of 20 flies per genotypeper time point). Page < 1 × 10−15, Pgenotype = 1.13 × 10−10, and Pinteraction =0.031 for ANCOVA. Error bars indicate ±SEM. (D) Kaplan–Meier survival curvesfor approximately 2-wk old female ppk28mutant (w,ppk28Δ) and backgroundcontrol (w) female flies [n = 149 (w); n = 150 (w,ppk28Δ)] under starvationconditions. (E and F ) Survival curves for female ppk28 deletion mutants(w,ppk28Δ) and background controls (w) [n = 245 (w); n = 247 (w,ppk28Δ);mean lifespan increase of 17.50 d (30.54%)] (E) compared with survival curvesrepresenting the addition of a genomic region containing the endogenousppk28 locus into both ppk28 mutant [w,ppk28Δ;+;Dp(320)] and control[w;+;Dp(320)] backgrounds (n = 180 [w;+;Dp(320)]; n = 137 [w,Δppk28;+;Dp(320)];mean lifespan increase of 1.14 d [1.65%]) (F) on SY5% (wt/vol) food. Pairwisecomparisons between statistically significantly different genotypes in survivalanalyses yielded P < 1 × 10−6 by log-rank test for all cases.

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gustatory signals negatively regulate Drosophila lifespan inde-pendently of feeding behavior and that loss of ppk28 functionis efficacious for sustained health and longevity.

ppk28-Mediated Signals Regulate Nutrient Homeostasis. The criti-cality of water for metabolic processes led us to hypothesize thatwater gustatory neurons may exert their regulatory effect throughcontrol of nutrient metabolism. To establish whether loss of ppk28input was sufficient to modulate nutrient homeostasis, we assayedwhole organism levels of both carbohydrate and lipid in the long-lived ppk28 deletion mutants. Indeed, these flies displayed bothincreased levels of triacylglyceride (TAG) and glucose comparedwith background controls, effects that were rescued with reintro-duction of ppk28 into the mutant background (Fig. 2 A and B).Notably, we observed that aging had little effect on TAG and glu-cose levels as differences among genotypes persisted throughoutlife. Although ppk28 mutants were slightly heavier than backgroundcontrols (Fig. S3A), normalization by body weight did not affect therelationship between genotypes (Fig. S3 B and C). Furthermore, thedifference in mass was not present immediately posteclosion (Fig.

S3D). These data suggest that altered nutrient levels in water-sensing mutants were due to a directed switch in physiological stateearly in adult life rather than a loss of homeostatic control.

ppk28-Mediated Lifespan Extension Requires Components of NutrientHomeostasis Pathways. Having observed significant differences innutrient levels, we next hypothesized that signaling pathwaysresponsible for the coordination of metabolic homeostasis maybe required for ppk28-mediated lifespan extension. In mammals,levels of glucagon and insulin are key to the coordination ofcarbohydrate and lipid metabolism (29). Flies maintain func-tionally homologous molecules to each of these hormones—theglucagon-like adipokinetic hormone (dAkh) (30) as well as eightinsulin-like peptides (dILPs 1–8) (31–33). To test the requirementof each signaling network in ppk28-mediated lifespan extension,we undertook an epistasis approach in which double mutant flies,functionally null for ppk28 as well as a critical component of eitherpathway, were generated and assessed for lifespan. We found thatthe extended lifespan of ppk28-null flies (Fig. 2C) was abrogatedby the introduction of a null mutation for AkhR (34) into thecontrol and ppk28 mutant backgrounds (Fig. 2D). Likewise, asimilar strategy using a null mutation for dFoxO (35), an in-tegral component in ILP signaling, also abolished the increasedlongevity found in ppk28 mutant flies (Fig. 2 E and F). Notably,this strategy does not allow genetically controlled comparisonsbetween AkhR and dFoxO mutants with w controls, and, as such,interpretation of lifespan effects of these single mutants is notwithin the scope of this study. As dFoxO is normally active underlevels of low ILP signaling, these data support a model by whichlifespan extension may be due, at least in part, to reduced levelsof insulin signaling. Consistent with this interpretation, we foundthat transcript levels of dILP2 were decreased in ppk28 mutants(Fig. S4A). Details of the downstream mechanism through whichdFoxO acts, however, remain unclear because whole-organismtranscript levels of dFoxO targets were variable (Fig. S4B).

ppk28 Signals Control Release of AKH from the Corpora Cardiaca. Therequirement of AKHR was a surprise as its cognate hormone hasbeen little studied in relation to aging. One prediction from thegenetic data is that release of AKH neuropeptide from its site ofsynthesis would be increased in ppk28 mutant flies. AKH is pro-duced in a small subset of neurons called “corpora cardiaca” (CC)(26). Although Akh mRNA levels were not significantly differentbetween ppk28mutant and control flies (Fig. S4C), previous work inboth the locust (Locusta migratoria) and Drosophila suggests thatAkh gene transcription is uncoupled from neuropeptide release andthat the neuropeptide may be discharged in a controlled fashionfrom a pool of continuously synthesized protein (36, 37). To de-termine whether loss of ppk28 affected AKH release or seques-tration, we directly imaged neuropeptide localization in dissectedCC from adult flies stained with a dAKH-specific antibody (26). Asa positive control indicative of active AKH signaling, we usedstarved control flies. AKH pathway activity is inversely correlatedwith starvation resistance (26), and starvation should thus stimulateAKH release. We found that ppk28 mutants showed increasedneuropeptide staining, compared with background controls,specifically in the axonal projections from which AKH is releasedto target areas. A complete lack of staining in these projectionswas never observed in ppk28 mutant animals but was frequentlyobserved in control animals (Fig. 3A and Fig. S5). The stainingpattern of ppk28 mutants closely resembled that of starved flies,arguing that loss of ppk28 function activates AKH signaling.

Activation of AKH-Producing Cells Extends Lifespan in Absence ofSensory Manipulation. Our results indicated that an increase inAKH signaling was induced in water-sensing mutants and wasessential for lifespan extension. These data are indicative ofAKH as a key effector of water taste sensation and as a cause of

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Fig. 2. ppk28 modulates lifespan through nutrient signaling. (A and B)Longitudinal measures of whole-fly TAG (A) and glucose (B) in ppk28 de-letion mutant female flies (w,ppk28Δ) and in mutant animals also containinga ppk28 genomic rescue construct [w,ppk28Δ;+;Dp(320)], as well as theirappropriate genetic background controls [w and w;+;Dp(320), respectively].n = 8–12 groups of five flies per genotype per time point. **P < 0.01; ***P <0.001 for the interaction term of two-way ANOVA. Error bars indicate ±SEM.(C and D) Kaplan–Meier survival curves for female ppk28 deletion mu-tant flies (w,ppk28Δ) and background controls (w) [n = 162 (w); n = 249(w,ppk28Δ); mean lifespan increase of 20.68 d (36.96%)] (C ) and thesame backgrounds containing loss of function of AKHR (w;AkhR−) and(w,ppk28Δ;AkhR–) [n = 253 (w;AkhR−); n = 248 (w,ppk28Δ;AkhR−); meanlifespan increase of 0.67 d (0.99%)] (D). (E and F ) Survival curves for fe-male ppk28 deletion mutant flies (w,ppk28Δ) and their controls (w) [n =221 (w); n = 243 (w,ppk28Δ); mean lifespan increase of 14.16 d (24.73%)](E ) and same backgrounds containing deletion of the FoxO transcriptionfactor (w;dFoxO− and w,ppk28Δ;dFoxO−) [n = 248 (w;dFoxO−); n = 80(w,ppk28Δ;dFoxO−); mean lifespan increase of 2.4 d (4.28%)] (F). Pairwisecomparisons between statistically significantly different genotypes yieldedP < 1 × 10−6 by log-rank test for all cases.

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ppk28 lifespan extension. If so, then increasing activity of thispathway, in the absence of sensory manipulation, should simi-larly increase lifespan. We therefore tested this hypothesis byconditionally expressing the activating cation channel dTRPA1(38) in Akh-ergic neurons and studying the effects of targetedneuronal activation on lifespan. Indeed, we found that transgenicflies (Akh-Gal4;UAS-dTRPA1) were significantly long-lived com-pared with genetic control animals at a temperature in whichdTRPA1 is activated (29 °C) but not at a control temperature(23 °C) in which it is not (Fig. 3B). Depleted TAG levels, which arecharacteristic of enhanced neuronal AKH secretion, persistedfor at least 26 d in flies with activated dTRPA1, suggesting thatthis genetic strategy was effective in securing chronic pathwaystimulation (Fig. S6A). Furthermore, this manipulation increasedstaining in CC cell axons, consistent with findings in long-livedppk28 mutants (Fig. S6B). Importantly, this lifespan extensioncould not be attributed to a reduction in food intake (Fig. S6C).The overexpression of Akh in Akh-ergic neurons (Akh-Gal4;UAS-Akh) had no effect on lifespan (Fig. S6D). This is not un-expected because of the documented uncoupling between AkhmRNA synthesis and secretion of AKH protein in insects wherephysiological phenotypes associated with activation of AKHsignaling, including an increase in larval hemolymph trehalose,are not recapitulated by modulation of gene overexpression(26). Together, these data suggest that neuronal modulation ofAkh-expressing cells is required to promote physiological changesthat are advantageous for maximizing health and longevity.

Loss of External Water Gustatory Information Induces Promotionof Internal Water Stores. The physiologic effects of ppk28 loss offunction were reminiscent of adaptations used by a number ofdesert species that have limited access to fresh water, which useTAG lipolysis and oxidation of free fatty acids as primary sourcesof metabolic water production (39, 40). We considered thatppk28 mutants, although not physically starved for water, mightnevertheless induce similar physiological strategies due to itsperceived scarcity. In this model, mutant flies should maintainhigher levels of internal water, which inDrosophila can be measuredby the subtraction of mass postdesiccation (“dry mass”) fromits initial mass (“wet mass”) (41). Indeed, ppk28mutant flies showedan increased change in mass after desiccation than backgroundcontrols (Fig. 4A). Although ppk28 mutants maintained a mod-est, yet significant, increase in wet mass (Fig. S3A), this is almostcertainly due to augmented stores of water, as well as TAG andglucose levels (Fig. 2 A and B), rather than an increase in grosssize. Indeed, whole-organism protein levels were not significantlydifferent between ppk28 mutants and their background controls(Fig. S7). Furthermore, insects with higher internal water con-tent have been found to be desiccation resistant (42), and wefound that ppk28 mutants exhibited significantly increased sur-vivorship under desiccating conditions (Fig. 4B). Consistent witha model by which internal water stores are increased through theaction of the AKH-signaling pathway, we found that constitutiveactivation of AKH-expressing cells also increased the differencebetween wet and dry mass (Fig. S8). Together, these results suggestthat loss of ppk28 function may drive the production of metabolicwater through a glucagon-like AKH-dependent mobilization of

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lipid stores as a compensatory response to the absence of sensoryinformation pertaining to accessible water.

DiscussionOrganisms are regularly subjected to diverse sensory stimuli,which provide vital information about the surrounding environ-ment. Establishing how individuals coordinate appropriate physi-ological responses to such cues is critical for understanding howa lifetime of perceptive experiences may affect overall health.Previous work has provided compelling evidence that nutrient-sensing signaling pathways are key regulators of longevity (10,43). Additionally, there is a conserved role for the function ofchemosensory systems in control of aging (1, 5, 6, 44, 45), in-cluding a study in PNAS showing that subsets of gustatory inputswere capable of modulating Drosophila lifespan in a bidirectionalmanner (6). One logical prediction followed that, in response totransduction and integration of a taste signal, an organism repro-grams its metabolic state through modulation of nutrient ho-meostatic pathways, ultimately altering lifespan. Which specificinputs—and the associated physiological states that result fromtheir manipulation—are capable of altering longevity, however,remained largely untested.Here, we exploited a specific gustatory signal—emanating from

water-sensing taste neurons—as a model by which to understandsensory modulation of aging. As water is an essential component ofan organism’s diet and its recognition and ingestion are critical toan organism’s health, we reasoned that modulation of this sensorycue alone may have significant physiological consequences. In-deed, we discovered that loss of function of the ion channel re-quired for water taste perception—ppk28—extended lifespan andaugmented health-related parameters (e.g., stress resistance andclimbing ability). Furthermore, mutation of ppk28 resulted inalteration of metabolic homeostasis through an increase in lipidstores and a subsequent activation of glucagon-like AKH sig-naling. Several observations suggested that this physiologicalswitch increased production of metabolic water, resemblinga strategy used by species with a severe or complete lack ofaccess to environmental water. Compatible with this model, wefind that activation of Akh-ergic neurons is sufficient to increaselifespan, suggesting that signals leading to such activation are alsoefficacious for increasing health and longevity (Fig. 4C).Although often overlooked as a principal dietary component

in favor of carbohydrate, protein, or lipid, water is just as crucialto an organism’s ability to maintain metabolic homeostasis andjust as quintessential for its survival. Indeed, diverse organisms,including flies and mammals, commit a similar amount of sen-sory resources to the perception of water (18, 46). Our studiessuggest that information about external water availability trans-duced through ppk28-sensing neurons is capable of affecting itsinternal production and that stimulation of this system by waterintake may represent an important consideration for determin-ing dietary influence on health status.Classic dietary restriction (DR) paradigms of carbohydrate

and protein robustly increase lifespan across species, and act atleast partially through sensory mechanisms independent of ca-loric intake (4, 44). Although conclusions from previous workover the role of dietary water in mediating the Drosophila DR–

lifespan extension axis have been mixed (24, 25), our studiessuggest that water restriction, inasmuch as it decreases stimula-tion of water-sensing neurons, may also be a viable strategy forenhancing physiological state. Given the necessity of water formetabolic reactions and the discomfort of thirst, however, ofperhaps more translational relevance is the implication that theglucagon-like AKH-signaling pathway is a potent modulator ofhealth and lifespan. There is some evidence suggesting that thispathway may, in fact, impinge on the control of lifespan viaprotein restriction as a recent study found that ectopic over-expression of Akh extends lifespan in flies which are fully fed, yet

not under yeast restriction (47). Although the ubiquitous natureof this manipulation may confound its physiological relevance,this is indicative that restriction of dietary components may con-verge on similar regulatory mechanisms. Congruously, levels ofplasma glucagon increase under dietary restriction in mice (48).Dietary or pharmacological interventions that stimulate release ofglucagon may, therefore, promote a healthier lifespan in mammals,without the necessity of actual restriction of dietary components.The evidence that ppk28-mediated inputs require the tran-

scription factor dFoxO for their lifespan effects suggests an addi-tional layer of complexity to this control, one potentially includingthe insulin/ILP-signaling pathway. Indeed, lifespan extension inC. elegans gustatory mutants requires the FoxO homolog daf-16(1), and work presented in PNAS suggests a similar dFoxO-dependent mechanism in D. melanogaster (6). The relationshipbetween the insulin and glucagon signaling pathways in main-taining metabolic homeostasis has been well studied in mamma-lian contexts, with high levels of insulin known to inhibit glucagonrelease (49). Although this association is less well understood inDrosophila, dILP2-producing median neurosecretory cells andAKH-producing cells are in close proximity, with dILP2-ergicaxons extending to the CC (50). If a similar cross-talk occurs inflies, activation of AKH release in ppk28mutants may be, possibly,in part due to down-regulation of ILP signaling, as suggested bydecreased dILP2 levels and dFoxO dependence of longevity in-crease in these flies. As such, lifespan modulation due to inter-ventions reducing insulin/ILP activity may additionally need to beunderstood in light of their effect on glucagon-like signaling.The significance of our findings for the enhancement of health and

longevity underscore the importance of the further work that remains.For instance, the mechanisms responsible for the initial increase inTAG stores used as substrate for AKH remain unknown. Further-more, the signal that activates AKH signaling in response to increasedTAG levels has also not been determined. Finally, it remains to bediscovered how an increase in AKH pathway activity is responsible forincreasing health and longevity. Nonetheless, the results describedhere form the basis for an understanding of the dynamics of lifespan-modulating sensory signals and their means of command.

Materials and MethodsFly Strains. For background controls, we used w1118-VDRC (Vienna DrosophilaRNAi Center) or yw (Bloomington Stock Center) lines. Transgenic flies includedw,ppk28Δ;+;+ (a gift from K. Scott, University of California, Berkeley, CA),w;+;Dp(1;3)DC320 (Bloomington Stock Center), w;UAS-dTRPA1;+ (a giftfrom P. Garrity, Brandeis University, Waltham, MA), w;AkhR−;+ (a gift fromR. Kühnlein, Max Planck Institute for Biophysical Chemistry, Göttingen,Germany), and w;+;dFoxO(Δ94) (a gift from L. Partridge, University CollegeLondon, London).

Survival Analyses. Lifespan analyses were performed using an empiricallyoptimized protocol established by our laboratory and facilitated by the useof an radiofrequency identification-based tracking system and associatedstatistical software (dLife) developed by our laboratory (51). See SI Materialsand Methods for technical details.

For desiccation resistance, approximately 2-wk-old flieswere placed in vialscontaining ∼1 cm drierite (anhydrous calcium sulfate; W. A. HammondDrierite Company) and allowed to desiccate at room temperature.

For starvation resistance, approximately 2-wk-old flies were placed in vialscontaining 1% agar and maintained at 25 °C with vials changed daily.

Nutrient Level Assays. For all nutrient measurements, female flies were frozenat −80 °C and homogenized in groups of five in 200 μL PBS + 0.05% TritonX-100 (IBI Scientific). Samples were centrifuged at 1,000 × g for 1 min tosettle debris. All reactions were read with Synergy2 plate reader (BioTek).See SI Materials and Methods for technical details.

Negative Geotaxis Assay. Flies maintained on SY10% food were placed inempty vials and forced to the bottom with four hard taps. Flies were allowedto climb for 2 s and then photographed. Images were analyzed using ClimberSoftware (developed by S.D.P.) to quantify the distance climbed.

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Immunohistochemistry. Adult female flies were reared as described thendissected at approximately 2 wk of age and stained with a dAKH-specificantibody (a gift from J. Park, University of Tennessee, Knoxville, TN), thenvisualized on Olympus FluoView 500 Laser Scanning Confocal Microscopeusing 20× magnification. Starved flies were placed on 1% agar vials for 14 hbefore dissection. See SI Materials and Methods for technical details.

Wet and Dry Mass Calculations. Groups of 10 female flies were weighed todetermine wet mass and then placed overnight at 65 °C and reweighed todetermine dry mass.

Statistical Methods. All survivorship data were compared via log-rank analysisbetween relevant genotypes. Analysis of covariance (ANCOVA) was used forthe negative geotaxis assay and a two-way ANOVA in analysis of TAG andglucose levels. Two-sided Student’s t tests were performed for wet/dry mass

calculations and quantitative PCR results. Sample sizes and replicate num-bers are explicitly stated in each figure.

ACKNOWLEDGMENTS. We thank many colleagues for the generous sharingof reagents. We also thank Jason Braco for technical advice on CC dissection,Alyson Sujkowski for instruction on the negative geotaxis assay, and MarkusNoll and Damian Brunner for use of laboratory space. Imaging wasperformed at the University of Michigan Microscopy and Image AnalysisLaboratory. This work was funded by US National Institutes of Health Grants5-T-32-GM007315 and T-32-AG000114 (to M.J.W.); T-32-AG000114 andF-32-AG042253 (to B.Y.C.); 5-T32-GM007863-32 (to Z.M.H.); R-01-AG030593,R-01-AG043972, and R-01-AG023166 (to S.D.P.); as well as the NovartisResearch Foundation (I.O. and J.A.), the Glenn Foundation, the AmericanFederation for Aging Research, and the Ellison Medical Foundation (S.D.P.).Additionally, this work used the Drosophila Aging Core of the Nathan ShockCenter of Excellence in the Biology of Aging funded by the National Instituteon Aging (P30-AG-013283).

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