Loss of tim or per extends lifespan in male Drosophila

To investigate how loss of specific circadian regulators influence aging, we examined the lifespans of four established arrhythmic Drosophila mutants (Fig. 1a): three genomic mutants, cycle (cyc01), period (per01), and timeless (tim01), and flies ubiquitously expressing a dominant-negative form of Clock (DN-Clock)21. Consistent with other reports3,4,5,6, functional disruption of the circadian transcriptional activators Cycle or Clock shortened the lifespan of male flies relative to controls (Fig. 1b, c). In contrast, functional disruption of the circadian transcriptional repressors Per and Tim significantly increased lifespan (15–20%) relative to controls; this appeared to be a male-specific effect (Fig. 1d, e). To confirm that lifespan extension was due to the loss of Per protein, we restored Per expression using the UAS–GAL4 system22. Expressing either of the two independent period transgenes using either the timeless–GAL4 driver or the ubiquitin–GAL4 driver in the per01 null background reverted per mutant lifespan to that of control animals (Supplementary Fig. 1A, B). Thus, loss of Per expression extends lifespan.

The classic method of lifespan extension is dietary restriction (DR). We showed previously that per01 and tim01 mutants are not diet-restricted—that is, these mutants eat more, not less, than controls (ref. 19; see also Fig. 2a). However, loss of Per and Tim might mimic physiological changes associated with DR. If so, DR should not further extend the lifespan of male per01 and tim01 mutants. In Drosophila, DR-mediated lifespan extension is accomplished by titration of protein (yeast extract (YE)). To test response to DR, we fed per01 and tim01 null mutants and controls four different concentrations of YE: 0.01% (low), 0.5% (DR), 3% (standard), and 10% (high). As we showed previously for female per01 and tim01 mutants10, male per01 and tim01 mutants exhibited DR-induced lifespan extensions similar to controls and lived longer than controls on most dietary protein concentrations (Fig. 1f, g). The lifespan of these circadian mutants was similar to that of control animals only at very high yeast concentrations (10%), which shortens lifespan. Thus, the extended longevity of per01 mutants appears to be independent of DR.

Fig. 2: period mutants exhibit high metabolic rate due to mitochondrial uncoupling. Relative to controls (gray), per01 mutants (green) exhibited: a increased feeding rate (n = 6 vials of ten flies/condition, p < 0.01); b decreased survival upon starvation (n ≥ 99 flies per condition, p < 0.001); c lower baseline levels of lipids (left) and increased rate of lipid utilization after 24 h of starvation (right), as shown by quantification of triacylglyceride (TAG) levels (n ≥ 4 samples/condition, 5 flies/sample, both p < 0.0001); d increased respiration, which was reverted by ubiquitous overexpression of Per during adulthood (n = 6 groups of 10 flies per condition); e higher CO 2 production over the circadian day (n = 6 groups of 10 flies/condition and timepoint); f higher respiration rates after 24 h of feeding with rotenone and oligomycin, but not with 2,4-DNP, or stearic acid (n ≥ 5 groups of 10 flies/condition); g increased oxygen consumption rate when stimulated through complexes I, II, and IV relative to controls (n = 4–6 oxygraph runs per condition); h increased leak respiration, using high-resolution respirometry on purified mitochondria (n = 5 oxygraph runs per condition, p < 0.001); i lower membrane potential, measured by JC-1 staining of purified mitochondria (n = 10 mitochondrial preps per condition, p < 0.001); and j faster recovery from cold shock (n = 26–30 flies per condition, p < 0.001). See Supplementary Information for n if not listed here; n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; p values were obtained by unpaired two-tailed t-test (a, c, e, g–i), ANOVA followed by Tukey’s post-hoc test (d, f), and log-rank analysis (b, j); error bars represent SEM. Full size image

We next tested if per01 mutants exhibit canonical changes in DR-associated mechanisms of longevity, including: decreased insulin signaling, measured by decreased phosphorylation of Akt protein; decreased TOR signaling, measured by decreased phosphorylation of S6K; and increased autophagy, measured by increased lipidation of Atg8. Surprisingly, per01 males were atypical for these hallmarks of longevity and, at some points in the circadian cycle, exhibited the opposite phenotype as predicted for long-lived mutants (Supplementary Fig. 1C). We further used genetic manipulations to determine if the inhibition of TOR signaling or the inhibition of insulin-like signaling (ILS) is responsible for the longevity of per01 mutants. To test the inhibition of TOR signaling, we ubiquitously suppressed TORC1 signaling in adulthood by inducible (RU486-mediated) overexpression of a dominant-negative form of the downstream kinase S6K, which is known to extend lifespan23. The inhibition of TORC1 signaling extended the lifespans of both control animals and per01 mutants to a similar magnitude (Fig. 1h), suggesting that the longevity of per01 mutants is independent of TORC1 inhibition. Feeding RU486 to either controls or per01 mutants lacking the UAS transgene had no influence on lifespan (Supplementary Fig. 1D). To test the inhibition of ILS, we performed partial genetic ablation of insulin-producing cells in the fly brain using the proapoptotic gene reaper24. This extended the lifespans of both control animals and per01 mutants to a similar magnitude (Fig. 1i), suggesting that per01-associated lifespan extension is independent of insulin-like signaling inhibition. Together, these results suggest that the longevity phenotype of per01 males is not due to canonical longevity mechanisms but due to a different, independent pathway.

per mutants exhibit a high metabolic rate

As metabolism and lifespan are linked, we set out to investigate the metabolism of long-lived per01 mutants. As shown in our previous work19, per01 males exhibited hyperphagia (increased feeding), decreased starvation resistance, low levels of lipid storage, and increased starvation-induced lipid utilization relative to controls (Fig. 2a–c, Supplementary Fig. 2A). Because per01 mutants eat more but are leaner than controls, we tested for hyperactive metabolic rate by measuring CO 2 production. Consistent with our previous characterization, per01 mutants produced more CO 2 throughout the circadian cycle relative to controls, which was reverted by exogenous Per expression (Fig. 2d, e); respiration rate was not affected in RU486 feeding controls (Supplementary Fig. 2B). Respiration rate is significantly affected by the mitochondrial function of oxidative phosphorylation. To determine if increased oxidative phosphorylation caused this increased metabolic output, flies were fed sublethal doses of mitochondrial complex inhibitors: rotenone, which blocks complex I of the mitochondrial electron transport chain (ETC), or oligomycin, which blocks complex V, the F 0 F 1 ATP synthase. While these compounds inhibited the CO 2 output of both control and per01 animals to a similar degree, per01 flies still had higher respiration rates than controls (Fig. 2f). This suggests that their increased respiration is independent of mitochondrial ATP synthesis and instead implicates a different mitochondrial function, such as mitochondrial uncoupling.

Mitochondrial uncoupling increases respiration by dissipation of the proton gradient and uncoupling of oxidative phosphorylation from ATP synthesis, creating futile cycles of respiration and generating heat, as in mammalian brown fat. To test the effects of mitochondrial uncoupling on respiration, we fed per01 mutants and control flies two different mitochondrial uncoupling compounds: 2,4-dinitrophenol (2,4-DNP), a proton ionophore that dissipates the proton gradient; and stearic acid, which induces mitochondrial uncoupling by activating endogenous UCP activity. Treatment with either drug increased the respiration of control flies to levels similar to those of per01 mutants but did not increase the respiration of per01 mutants (Fig. 2f). These results suggest that the increased respiration of per01 mutants is due to increased mitochondrial uncoupling and that per01 mutants may already be maximally uncoupled within viable parameters. Higher doses of uncoupling drugs were lethal to both genotypes.

To directly test whether per01 mutants were mitochondrially uncoupled relative to controls, we assessed mitochondrial function in vitro. We purified mitochondria from per01 mutants and controls and measured O 2 consumption. per01 mutant mitochondria exhibited increased O 2 consumption when initiated through ETC complexes I, II, and IV (Fig. 2g). This increased O 2 consumption was not due to increased mitochondrial ETC protein abundance or increased enzymatic activity (Supplementary Fig. 2C, D). Instead, per01 mitochondria exhibited two critical hallmarks of mitochondrial uncoupling relative to control mitochondria: increased leak respiration, measured by higher oxygen consumption after oligomycin treatment (Fig. 2h); and decreased membrane potential, or disrupted proton gradient, measured by the membrane potential dye sensor JC-1, both during steady-state ATP generation and after inhibition by oligomycin (Fig. 2i). Finally, to assess heat generation, another hallmark of mitochondrial uncoupling, we performed cold shock recovery assays on per01 mutants and control animals (Fig. 2j). After 1 h of cold shock at 4 °C, per01 mutants recovered significantly faster than controls, suggesting that per01 mutants may generate more heat than controls. Thus, loss of Per protein increases mitochondrial uncoupling.

Uncoupling protein UCP4C is required for per mutant longevity

To determine if this increased mitochondrial uncoupling is required for the lifespan extension of per01 mutants, we genetically manipulated the expression of endogenous proteins that cause mitochondrial uncoupling. Like many animals, Drosophila can undergo mitochondrial uncoupling by induction of UCPs, including UCP4A, B, and C25,26. Of these, we found that the expression of Ucp4B and Ucp4C is circadian-regulated in control flies and constitutively high in per01 mutants (Fig. 3a–c). To test the role of UCPs in the metabolic phenotype observed in per01 mutants, we first disrupted the expression of both UCP4B/C proteins using a mutant containing a piggyBac transposon in the intergenic region between these two closely linked Ucp4 genes (Supplementary Fig. 3A, B). Next, to determine if period nulls exhibit true mitochondrial uncoupling via classic mitochondrial UCPs, we tested stimulation of mitochondrial respiration in the presence of palmitate and reversal by the UCP inhibitor guanosine nucleotide (GTP) suppression of respiration. Palmitate and GTP are respectively known to stimulate and suppress classic mitochondrial UCPs27,28,29. Indeed, per01 flies showed significantly increased respiration in the presence of palmitate, which was reversed by the addition of GTP (Supplementary Fig. 3C). per01 flies with disrupted UCP4B/C expression showed no response to palmitate or GTP indicating that the uncoupled respiration is due to UCP gene function. In addition, disruption of UCP4B/C in per01 flies reverted other hallmarks of uncoupling: mitochondrial leak respiration (Fig. 3d); mitochondrial membrane potential (Fig. 3e); mitochondrial oxygen consumption rate (Supplementary Fig. 3D); and whole-animal cold shock recovery rates (Fig. 3f). Disruption of the UCP4B/C expression not only reverted mitochondrial uncoupling but also reverted per01 lifespan to that of controls, suggesting that mitochondrial uncoupling causes lifespan extension (Fig. 3g). To inhibit UCPs by an orthogonal mechanism, we performed RNAi-mediated knockdown of UCP4A, B, and C in adulthood throughout the whole body in both per01 mutants and controls. Consistent with our mutant analysis, knockdown of UCP4B or UCP4C, but not UCP4A, reverted per01 lifespan to that of control animals (Supplementary Fig. 3E–G). Thus, the Ucp4B/C expression is necessary for the longevity and metabolic phenotypes of per01 mutants.

Fig. 3: UCP4C is necessary for period mutant lifespan and sufficient to extend wild-type lifespan. Relative to controls (gray), per01 mutants (green) exhibited: a higher expression of Ucp4B and Ucp4C but not Ucp4A; and constitutively high expression of b Ucp4B and c Ucp4C, both of which are circadian-regulated in controls. Relative to controls (gray), per01 mutants (green) also exhibited the following phenotypes, which were reverted by suppression of Ucp4B/C expression, comparing flies with (dashed lines) or without (solid lines) piggyback mutation of Ucp4B/C: d increased leak respiration by purified mitochondria; e decreased mitochondrial membrane potential; f faster cold shock recovery; and g increased lifespan. Relative to vehicle-fed controls (gray), daGS > UAS-Ucp4C flies fed RU486 to induce constitutive UCP4C overexpression (magenta) exhibited: h higher leak respiration (p < 0.001); i lower mitochondrial membrane potential (p < 0.001); and j increased lifespan (p < 0.0001). k Ubiquitous overexpression of UCP4C in otherwise wild-type flies was sufficient to extend lifespan (gray, dashed) relative to driver-only controls (gray, solid). In per01 mutants (green, solid), overexpression of UCP4C did not further extend the lifespan of per01 mutants (green, dashed). See Supplementary Table 1 for n and statistical analysis of lifespans, particularly multicurve comparisons; n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; p values were obtained by unpaired two-tailed t-test (a, h, i), ANOVA followed by Tukey’s post-hoc test (d, e), and log-rank analysis (f, g, j, k); error bars represent SEM. Full size image

Finally, to determine if increased UCP expression is sufficient to extend the lifespan of WT animals, we ubiquitously overexpressed UCP4C at low levels during adulthood using conditional GeneSwitch drivers induced by the drug RU486. Flies overexpressing UCP4C showed mitochondrial uncoupling phenotypes very similar to per01 mutants; increased mitochondrial leak respiration (Fig. 3h), decreased mitochondrial membrane potential (Fig. 3i), increased CO 2 production (Supplementary Fig. 4A), and faster cold shock recovery (Supplementary Fig. 4B). Most importantly, the constitutive expression of UCP4C extended the lifespan of otherwise wild-type flies to the same extent as per01 mutants, with no effect on per01 mutants (Fig. 3j–k). Feeding these low doses of RU486 alone had no effect on either metabolic or lifespan phenotypes when flies lacked the UAS transgene (Supplementary Fig. 3C–G). These results suggest that UCP4C functions in the same pathway as Per to extend per01 mutant lifespan and that increased expression of the mitochondrial uncoupling protein UCP4C extends lifespan. Taken together, our data point to mitochondrial uncoupling as the major circadian-regulated physiology directly responsible for the extended lifespan of per01 mutants.

Lifespan extension is mediated by loss of Per in intestines

While circadian sleep/wake cycles are coordinated by a neuronal master clock in the brain, many other circadian functions are regulated by peripheral clocks in specific tissues30. To determine whether the master clock or a peripheral circadian clock mediates per01 longevity, we rescued period expression in different organ systems via the UAS–GAL4 system. While ubiquitous expression of Per protein during adulthood reverted the lifespan of per01 mutants to that of controls (Fig. 4a), neuronal expression of Per did not (Fig. 4b). In contrast, intestinal expression of Per in the whole intestine or specifically in intestinal stem cells (ISCs) and enteroblasts (EBs) reverted per01 lifespan to that of controls (Fig. 4c–e). This result suggests that loss of Per in the intestine is required for lifespan extension of per01 mutants.

Fig. 4: Lifespan extension is mediated by loss of Per specifically in the intestine. Tissue-specific rescue of Per expression (dashed lines) in the per01 background (green) and controls (gray). a Ubiquitous rescue of Per during adulthood was sufficient to revert per01 lifespan to control levels. While neuronal overexpression of Per (b) during adulthood did not revert per01 lifespan, intestinal overexpression of Per (c, d) was sufficient to revert per01 lifespan to control levels. e Rescue of Per specifically in intestinal stem cells (ISCs) and enteroblasts (EBs) during development or adulthood reverted per01 lifespan to control levels. f Loss of period through ubiquitous CRISPR-mediated deletion during adulthood extended lifespan of otherwise wild-type flies, with no further lifespan extension in per01 nulls. CRISPR-mediated deletion of period in g the intestine or h IScs and EBs also extended lifespan. See Supplementary Table 1 for n and p values for lifespan experiments, particularly multicurve comparisons; p values were obtained by log-rank analysis. Full size image

To directly test if loss of per in the intestine is sufficient to extend lifespan, we used a UAS–GAL4-based CRISPR system to disrupt the period gene31,32. Ubiquitous deletion of per via the daughterless GeneSwitch driver (daGS–Gal4) was sufficient to extend lifespan of control flies but not per01 mutants (Fig. 4f). Similar to per01 mutants, flies with ubiquitous disruption of period were completely arrhythmic in constant darkness (Supplementary Fig. 5A). We then performed tissue-specific CRISPR-mediated disruption of period in either the whole intestine or specifically in ISCs/EBs during either development or adulthood and found that any of these manipulations was sufficient to extend lifespan (Fig. 4g, h). Disruption of period in the whole intestine or ISCs and EBs had no influence on circadian locomotor activity (Supplementary Fig. 5B, C). To control for CRISPR-mediated DNA damage, we disrupted an unrelated gene involved in sperm storage in females (acp98AB)33 using the same inducible GAL4 drivers. acp98AB disruption did not extend lifespan using any of these tissue-specific drivers (Supplementary Fig. 5D–F). Similarly, RU486 feeding had no influence on control and per01 mutants lacking UAS transgenes (Supplementary Fig. 5G–I). CRISPR-targeted disruption of period in ISCs/EBs resulted in >90% reduction in per transcript in the intestine after 30 days of induction (Supplementary Fig. 5J). Taken together, these data show that the intestinal circadian clock plays a role in limiting lifespan in Drosophila.

UCP4C in the intestine is necessary for per mutant longevity

To determine if mitochondrial uncoupling is circadian-regulated in the intestine, we first measured mRNA levels of Ucp4C in dissected whole intestines during the day and night (Fig. 5a). In control intestines, Ucp4C was low during the day and elevated at night, while per01 mutants exhibited constitutively high Ucp4C expression during both day and night. To further evaluate circadian regulation of uncoupling activity in the gut, we assessed mitochondrial membrane potential at different times of day in dissected intestines from wild-type flies and per01 mutants, stained with the dye TMRE, which accumulates in mitochondria with higher membrane potential. Loss of membrane potential indicates increased mitochondrial uncoupling. In controls, intestinal mitochondrial membrane potential was indeed circadian-regulated, with higher membrane potential (lower uncoupling) during the day and lower membrane potential (higher uncoupling) at night (Fig. 5b, c). Consistent with loss of circadian regulation, per01 mutants exhibited low mitochondrial membrane potential at both timepoints, suggesting high levels of mitochondrial uncoupling during both day and night. This phenotype is not due to changes in mitochondrial abundance, as both controls and per01 mutants have similar intestinal mitochondrial populations, as measured by mitoGFP fluorescence (Supplementary Fig. 6A, B). Thus, mitochondrial uncoupling in the Drosophila intestine is circadian-regulated.

Fig. 5: UCP4C in the intestine is necessary for loss of Per-mediated lifespan extension and sufficient to extend wild-type lifespan. a Whole intestine expression levels of Ucp4C during day and night show oscillations of Ucp4C expression in controls with constitutively high expression in per01 mutants. b Representative images of the posterior midgut of control (left) and per01 mutants (right) stained with Hoechst (DNA) and TMRE during the day and night (scale bar = 30 μm). c While wild-type intestines exhibited high membrane potential during the day and low membrane potential at night, per01 flies exhibited low membrane potential at both times of day. Knockdown of Ucp4C in the whole intestine (d) or ISCs/EBs (e) reverted per01 lifespan to control levels. Relative to controls (gray), flies overexpressing Ucp4C (magenta) in the whole intestine (f) or ISCs and EBs (g, h) had extended lifespan (p < 0.0001 for each). See Supplementary Table 1 for n and statistical analysis of lifespans; n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; p values were obtained by ANOVA followed by Tukey’s post-hoc test (a, c) and log-rank analysis (d–h); error bars represent SEM. Full size image

Because we found that ubiquitous UCP4C expression was necessary and sufficient for lifespan extension (Fig. 3), we next tested whether intestine-specific UCP4C expression is necessary and sufficient for lifespan extension. Similar to per rescue, RNAi-mediated knockdown of UCP4C in the whole intestine or in ISC/EB populations reverted per01 lifespan to that of controls (Fig. 5d, e). Moreover, while the overexpression of UCP4C in the nervous system did not alter lifespan (Supplementary Fig. 6C), constitutive expression of UCP4C in the whole intestine (Fig. 5f, Supplementary Fig. 6D) or specifically in ISCs and EBs (Fig. 5g, h) resulted in extended lifespan and lowered mitochondrial membrane potential in otherwise wild-type animals (Supplementary Fig. 6E–H). Thus, intestinal UCP4C expression is not only necessary for the lifespan extension of per01 mutants but also sufficient to extend lifespan in otherwise wild-type Drosophila. That is, intestine-specific upregulation of a normally circadian-regulated, oscillating physiological function, mitochondrial uncoupling, extends lifespan.

Per and UCP4C control intestinal homeostasis via ROS levels

To understand the underlying mechanism by which mitochondrial uncoupling in the intestine extends lifespan, we tested if loss of per or intestinal UCP4C overexpression delayed aging-related defects in the intestine. When flies age, they typically lose intestinal barrier function, which is also a major predictor of mortality34,35,36,37, suggesting that lifespan can be extended by preventing aging-related intestinal barrier dysfunction. We tested for aging-related intestinal barrier dysfunction using the “smurf assay,” named after a children’s cartoon, which measures leakage of an ingested blue dye36,37. Consistent with their extended lifespan, old per01 mutants showed a lower percentage of “smurfs” relative to controls, indicating a delay in aging-related intestinal barrier dysfunction (Fig. 6a). Moreover, this maintenance of intestinal integrity observed in per01 mutants depended on intestinal expression of UCP4C and intestinal overexpression of UCP4C alone in otherwise wild-type flies was sufficient to maintain intestinal integrity. Thus, loss of Per and increased mitochondrial uncoupling in the intestine protect against aging-related intestinal dysfunction.

Fig. 6: Loss of period preserves intestinal homeostasis via increased mitochondrial uncoupling and decreased ROS levels. a Smurf assays of 45-day-old flies showed per01 mutants had reduced populations with intestinal barrier dysfunction, a phenotype dependent upon Ucp4C expression, and that overexpression of Ucp4C also reduced intestinal barrier dysfunction. b Quantification of intestinal pHH3+ staining showed that per01 mutants had lower levels of age-related hyperproliferation dependent on UCP4C expression and that overexpression of UCP4C caused a large reduction in mitotic cells (n = 13–17 intestines). c Representative images of phospho-histone H3 at residue S10 (pHH3) staining of midguts from 45-day flies (scale bar = 40 μm). (d, e) per01 mutants and Ucp4C-overexpressing flies exhibited delayed esg + cell overproliferation (e) and lower ROS output of all posterior midgut cells, including ISC/EB populations. f Representative images for MitoSOX staining of aged posterior midguts in control esg-GAL4 > GFP flies, per01; esg-GAL4 > GFP, and esg-GAL4 > GFP;UAS-Ucp4C flies (scale bars = 35 μm (top) and 15 μm (bottom inset location indicated by dashed lines). See Supplementary Table 1 for n and statistical analysis of lifespans; n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; p values were obtained by ANOVA followed by Tukey’s post-hoc test (b, c, e, f); error bars represent SEM. Full size image

As aging-related intestinal barrier dysfunction is linked to overproliferation and tissue dysplasia in the gut38, we next tested if loss of per or intestinal UCP4C overexpression delayed aging-related cellular proliferation in the gut. Per protein has previously been shown to regulate ISC regeneration during acute intestinal stress39. To measure cellular proliferation in the intestines, we stained aged per01 and control flies for phospho-histone H3 at serine 10 (pHH3), a standard proliferative marker that labels mitotic cells (Fig. 6b, c). Further consistent with delayed aging, per01 mutants showed fewer pHH3 positive cells in the gut relative to control flies. To determine if this phenotype depends on mitochondrial uncoupling, we knocked down UCP4C in per01 flies and controls. UCP4C knockdown reverted the number of proliferative cells in per01 flies to the higher levels seen in controls. Moreover, the overexpression of UCP4C in otherwise wild-type controls was sufficient to decrease age-related overproliferation of ISCs/EBs. Thus, similar to the smurf assay and consistent with lifespan results, intestinal UCP4C expression is both necessary in per01 mutants and sufficient in otherwise wild-type controls to suppress aging-related intestinal cellular proliferation.

Because the microbiome and age-related intestinal dysbiosis can contribute to intestinal barrier dysfunction, intestinal dysplasia, and proliferative state40,41,42, we sought to determine if gut bacteria play a role in period null longevity. First, we measured overall microbial load in aging flies using universal 16S bacterial qPCR. Higher bacterial load is associated with aging and mortality. We found that long-lived per01 flies did not have decreased bacterial load but instead exhibited an increased bacterial load during aging relative to controls (Supplementary Fig. 7A). To determine if this increased microbial load influenced lifespan, we fed control and per01 flies throughout adulthood an antibiotic cocktail that reduced intestinal bacterial levels to nearly undetectable levels (Supplementary Fig. 7B). Feeding antibiotics for the entire lifespan had no impact on the extended longevity of per nulls relative to controls (Supplementary Fig. 7C). Together, these data show that the microbiome present in our animals did not cause per01 longevity.

Intestinal dysfunction and increased aging-related cellular proliferation are closely associated with elevated Reactive Oxygen Species (ROS) production. ROS can serve as important mitogenic signals in stem cells and their differentiated progeny cells in the fly43; elevated ROS production in the Drosophila intestine is thought to lead to increased misdifferentiation of proliferative cells, ISCs, and EBs44. Because mitochondrial uncoupling can restrict ROS output45,46,47, we hypothesized that uncoupled per01 mutants have extended longevity due to decreased mitochondrial ROS production in the intestine, leading to a decrease in age-related overproliferation of esg-positive (esg+) ISC/EB cells. To test this, we stained the intestines of aged per01 mutants, flies expressing UCP4C constitutively in ISCs and EBs, and controls with MitoSOX Red, a fluorescent mitochondrial ROS (superoxide) indicator (Fig. 6f). Both per01 mutants and intestinal UCP4C-expressing flies exhibited reduced esg+ populations and decreased mitochondrial superoxide production relative to controls in the whole posterior midgut, as well as in ISC/EB populations (Fig. 6d, e). Together, these results suggest that loss of Per function and increased mitochondrial uncoupling via upregulated UCP4C decrease ROS levels in the intestine.

Uncoupling drugs extend lifespan via gut homeostasis

To extend our genetic experiments, we tested if pharmacological induction of mitochondrial uncoupling recapitulates the intestinal phenotypes of per01 mutants and extends lifespan. We tested low dietary concentrations of two compounds, the mitochondrial uncoupling compound 2,4-DNP and uncoupling-agent/antioxidant beta-hydroxytoluene (BHT). To identify the effective dose of these compounds, we first showed that both compounds extended the lifespan of wild-type male flies when used within a range of physiological concentrations (Fig. 7a–c, Supplementary Fig. 6A)48,49. To ensure that these drugs did not cause diet restriction, we confirmed that DNP or BHT did not decrease feeding rate (Supplementary Fig. 6B). We found that these drugs did not extend the lifespan of per01 mutants, suggesting that these mutants are already receiving the maximal benefit of mitochondrial uncoupling (Supplementary Fig. 6C, D). Consistent with the hypothesis that uncoupling-induced lifespan extension is mediated by reduced cellular proliferation and decreased ROS levels, dietary DNP at concentrations that extend lifespan also reduce age-related esg+ cell overproliferation and mitochondrial ROS output (Fig. 7d, e). Thus, pharmacological induction of mitochondrial uncoupling extends lifespan and recapitulates many of the metabolic phenotypes of per01 mutants and UCP4C-expressing flies.

Fig. 7: Pharmacological reduction of ROS via uncoupling preserves intestinal homeostasis and extends lifespan. Flies fed the mitochondrial uncoupler 2,4-DNP (magenta) either throughout their lifespan (a) or only during adulthood (b) showed extended lifespan relative to vehicle controls (gray, p < 0.0001 for each). c 2,4-DNP feeding increased median lifespan in a dose-dependent manner. d Representative images of MitoSOX staining of posterior midguts in esg-GAL4 > GFP flies fed vehicle or DNP (scale bars = 25 μm, top; 10 μm, bottom, inset location indicated by dashed lines). e Flies fed 2,4-DNP exhibited: fewer esg + cells and thus lower ISC/EB overproliferation with age (left); decreased Mitosox staining of the entire posterior midgut and thus decreased mitochondrial superoxide output (middle); and decreased MitoSOX staining specifically in ISCs/EBs marked by GFP (right). f Representative images of midguts from flies exhibiting loss of Notch-mediated tumor formation (scale bar = 35 μm). Relative to vehicle-fed flies, flies with Notch-induced tumor formation that were fed 2,4-DNP exhibited delayed tumor formation as measured by GFP + area in the midgut (g) and extended lifespans (h, p < 0.0001 for each). See Supplementary Table 1 for n and statistical analysis of lifespans; n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; p values were obtained by log-rank analysis (a, b, c, h), unpaired two-tailed t-test (e), and ANOVA followed by Tukey’s post-hoc test (g); error bars represent SEM. Full size image

To test whether the mitochondrial uncoupling-induced suppression of cellular overproliferation is specific to aging-related cellular overproliferation and misdifferentiation or may act as a general mechanism to suppress cellular overproliferation, we tested the effects of the uncoupling drug DNP on an induced intestinal tumor model. Notch-Delta signaling normally keeps proliferative intestinal cells (ISCs/EBs) at a homeostatic level of differentiation and division50; loss of Notch in ISCs and EBs causes cellular overproliferation and the formation of ISC-derived intestinal tumors50,51. Changes to ISCs that alter differentiation and result in overproliferation are a common step in cancer development52. To test if mitochondrial uncoupling could slow tumor progression in the gut, we induced ISC tumors by RNAi suppression of Notch signaling, followed by feeding of 2,4-DNP (to induce mitochondrial uncoupling) or vehicle. Dietary 2,4-DNP slowed the progression of ISC tumors (Fig. 7f, g) and extended the short lifespan of these animals (Fig. 7h). These results suggest that pharmacological induction of mitochondrial uncoupling can metabolically suppress stem cell proliferation not only during normal aging but also during tumorigenesis.