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Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids

Subjects

Abstract

Dietary restriction (DR) promotes healthy aging in diverse species. Essential amino acids play a key role, but the molecular mechanisms are unknown. The evolutionarily conserved Sestrin protein, an inhibitor of activity of the target of rapamycin complex 1 (TORC1), has recently been discovered as a sensor of amino acids in vitro. Here, we show that Sestrin null mutant flies have a blunted response of lifespan to DR. A mutant Sestrin fly line, with blocked amino acid binding and TORC1 activation, showed delayed development, reduced fecundity, extended lifespan and protection against lifespan-shortening, high-protein diets. Sestrin mediated reduced intestinal stem cell activity and gut cell turnover from DR, and stem cell proliferation in response to dietary amino acids, by regulating the TOR pathway and autophagy. Sestrin expression in intestinal stem cells was sufficient to maintain gut homeostasis and extend lifespan. Sestrin is thus a molecular link between dietary amino acids, stem cell function and longevity.

Main

Dietary restriction (DR) can extend lifespan in diverse species, including fruit flies and rodents1,2. DR not only increases longevity but also induces broad health improvements during aging in rodents, primates and humans3,4,5,6. Restriction of specific dietary components, especially protein, is more important than the overall reduction in calories in producing the responses to DR7,8,9,10,11. Furthermore, restriction of specific essential amino acids, including methionine12,13 and the branched-chain amino acids (BCAAs) leucine, isoleucine and valine14,15,16, can extend lifespan. However, the underlying molecular and cellular mechanisms are still elusive.

DR maintains the health of multiple tissues, including brain, bone, muscle, gut and fat17,18,19. However, their contributions to DR-mediated longevity are currently unclear. In Drosophila, the gut is a key mediator20. This tissue consists of self-renewable intestinal stem cells (ISCs), transient enteroblasts, and differentiated absorptive enterocytes and enteroendocrine cells21,22. Interestingly, both ISCs and enterocytes contribute to fly longevity23,24. Furthermore, both the geroprotective target of rapamycin complex 1 (TORC1) inhibitor rapamycin25 and amino acid restriction14 improve gut health. However, the molecular mechanisms at work are currently unknown.

Reduced activity of the nutrient-sensing insulin/insulin-like growth factor (IGF-1)/TOR (IIT) network can extend lifespan across model organisms1,2, and it plays a key role in the responses to DR, with the longevity of flies with reduced IIT signalling unresponsive to DR11,26,27. The IIT network monitors and integrates different environmental cues including amino acids28,29, which mainly affect the TOR branch of the network. Recently, Sestrin proteins have been shown to act as upstream negative regulators of TORC1 activity in vitro in response to amino acid availability30. Sestrins are highly conserved proteins that are induced in cells under environmental challenges, including oxidative stress, DNA damage and amino acid starvation31. Mammalian genomes encode three Sestrin proteins, while the Drosophila genome encodes a single Sestrin orthologue. Sestrin proteins inhibit TORC1 activity, both through activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and through interaction with the GTPase-accelerating protein (GAP) activity towards Rags (GATOR) complex30,32. Sestrin directly binds to amino acids, resulting in the dissociation of the Sestrin–GATOR2 complex and increased TORC1 activity33,34. On the basis of these in vitro studies, Sestrin has been postulated to act as a leucine sensor33,34. However, whether Sestrin performs similar functions in vivo is not known35. In Drosophila, loss of Sestrin causes age-associated pathologies, probably through chronic TORC1 activation and hence reduced autophagy36. Surprisingly, however, Sestrin mutants were reported to have normal lifespan31. Thus, the in vivo roles of Sestrin remain to be determined.

Here we uncovered roles of amino acid sensing by Sestrin in whole organism physiology in vivo. Loss of Sestrin blunted the normal increase in lifespan in response to DR. We identified a key role of Arg407 in the fly Sestrin protein for amino acid binding in vitro. We generated a Sestrin mutant fly line (SesnR407A) in which amino acid binding to the endogenous Sestrin protein was blocked, which prevented activation of TORC1 by amino acids in vivo. SesnR407A mutant flies showed reduced TORC1 activity, decreased growth, delayed development, increased lifespan and improved gut homeostasis. Adding back amino acids to DR food medium increased ISC proliferation in control flies, but this effect was blocked in SesnR407A mutant flies. Sestrin regulated lifespan and ISC maintenance via the TOR pathway, and autophagy in ISCs was essential to regulate gut cell turnover. Furthermore, Sestrin expression in ISCs was sufficient to maintain gut homeostasis and increase fly lifespan, establishing Sestrin as a novel molecular link that regulates stem cell proliferation, intestinal health and longevity in response to dietary amino acids.

Results

Absence of Sestrin attenuates the response of lifespan to dietary restriction

To determine the role of Sestrin in amino acid sensing in vivo, we used a previously generated Sestrin mutant allele36, termed Sesn3F6, in which the first two exons of the Sestrin gene are deleted (Extended Data Fig. 1a). Sesn3F6 mutant flies had greatly reduced Sestrin messenger RNA levels and no detectable Sestrin protein (Extended Data Fig. 1b,c), suggesting it to be a strong hypomorph or even a functionally null allele. The Sesn3F6 allele was backcrossed into the white Dahomey (wDah) wild-type strain and wDah flies were used as controls in subsequent experiments. We examined the effect of loss of Sestrin on whole organism physiology by measuring development time. Sesn3F6 mutant flies were viable and had more rapid egg-to-adult development and slightly increased body weight compared with controls (Extended Data Fig. 1d,e), consistent with increased TORC1 activity during larval development, as previously reported36. Loss of Sestrin therefore increased larval growth rate and adult body size.

We next assessed the in vivo role of Sestrin in the adult fly. In Drosophila, increased lifespan from DR is mediated almost exclusively by restriction of essential amino acids in the diet11. We therefore measured the response of lifespan to DR in Sesn3F6 mutant flies. In Drosophila, DR can be achieved by diluting the yeast component of the diet, and we measured survival of female flies on four different food dilutions: 0.1×, 0.5×, 1.0× and 2.0× the yeast content of the normal sugar-yeast-agar (SYA) fly diet. wDah females showed a typical tent-shaped response of lifespan to DR, with the longest median lifespan on the 0.5× and 1.0× yeast foods (Fig. 1a,b). Interestingly, Sesn3F6 mutant flies showed an attenuated increase in lifespan at these two yeast concentrations, with median lifespan compared with controls reduced by 11% and 13% on the 0.5× and 1.0× yeast food, respectively (Fig. 1a,b). However, the mutants had similar lifespan to controls at high and low yeast concentrations, showing that they were normally viable and that their attenuated lifespan was specific to DR conditions. Cox proportional hazard (CPH) analysis confirmed that the interaction between genotype and treatment was significant (P < 0.0001), demonstrating that Sesn3F6 mutant flies responded to the DR treatment differently from controls. Thus, in Drosophila, although parallel systems also mediate the response, Sestrin function is essential to achieve full lifespan extension in response to DR.

Fig. 1: Loss of Sestrin attenuates the response of lifespan to DR.
figure1

a,b, Survival (a) and median lifespan (b) of wDah and Sesn3F6 females under DR. On 0.1× food and 2.0× SYA food, Sesn3F6 flies showed similar lifespan to wDah, with P = 0.10 and P = 0.18, respectively, log-rank test. On the 0.5× and 1.0× DR food, the lifespan of Sesn3F6 mutants was significantly reduced, with a decrease in median lifespan of 11% and 13%, respectively. P = 4.4 × 10−5 (0.5× food), P = 4.8 × 10−14 (1.0× food), log-rank test. Sesn3F6 flies responded to DR food significantly differently from wDah, P < 0.0001, CPH analysis; n = 100 flies for each condition of each genotype. The lifespan assays were repeated twice.

Source data

Generation of an amino acid-sensing defective Sestrin fly mutant

Sestrin activity towards TORC1 in vitro is directly regulated by its binding of amino acids33. To determine whether the amino acid-sensing ability of Sestrin is important for its function in vivo, we generated a novel fly strain in which the putative amino acid binding pocket of the Drosophila Sestrin protein was mutated. In the human Sestrin2 protein, arginine residue 390 (R390) is an essential component of the binding pocket, and mutation of R390 to alanine completely abolishes leucine binding34. By sequence homology analysis, we identified arginine 407 (R407) as the corresponding residue in the Drosophila Sestrin protein (Fig. 2a). Modelling the structure of the Drosophila Sestrin based on the structure of the human protein showed that R407 is indeed located in a pocket-like structure (Fig. 2b), suggesting that R407 might be a key residue for amino acid binding in the fly Sestrin protein. To test this, we performed Sestrin–GATOR2 interaction assays in vitro33 (Fig. 2c,d). A hemagglutinin (HA)-tagged Drosophila Sestrin wild-type or R407A mutant protein was co-expressed with the fly Flag-tagged WD repeat domain 24 (WDR24) protein in HEK-293T cells. Individual amino acids were added to the cell lysate followed by Flag immunoprecipitation of the WDR24 protein. Addition of leucine, isoleucine, valine and methionine interfered with the Sestrin–GATOR2 interaction of the wild-type Sestrin protein, but they did not affect the interaction of the Sestrin R407A mutant protein (Fig. 2c,d). The R407 residue thus plays a key role in the amino acid binding of the Drosophila Sestrin protein. To analyse the in vivo impact of amino acid sensing by Sestrin on fly physiology, we employed clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) to introduce the R407A mutation into the endogenous Sestrin gene locus (Fig. 2e). In addition, we introduced a Myc tag before the TGA stop codon of the Sestrin gene to allow easy detection of the protein by immunoblotting, and the resulting fly line was termed SesnR407A. To control for the presence of the Myc tag, we generated an additional fly strain termed Sesnwt, which only contained the Myc tag. We next used quantitative PCR with reverse transcription (RT–qPCR) and immunoblotting analysis to show that the introduced SesnR407A mutation did not affect Sestrin mRNA or protein levels, respectively (Extended Data Fig. 2a–c).

Fig. 2: Generation of an amino acid-sensing defective Sestrin fly mutant.
figure2

a, Partial sequence alignment of Sestrin proteins including the arginine (R) residue crucial for leucine binding (red). Sestrin2 from H. sapiens and M. musculus were used for the alignment. b, Structure of the Drosophila Sestrin protein modelled by UCSF Chimera on the human Sestrin2 protein (PDB 5DJ4). The R407 residue (highlighted in red) is located in a pocket. c,d, Sestrin–GATOR2 in vitro interaction assays using the Drosophila wild-type Sestrin protein and the Drosophila Sestrin R407A mutant protein. Flag immunoprecipitation (IP) was performed followed by immunoblotting against the HA and Flag tag, respectively. c, BCAAs and methionine interfered with Sestrin–GATOR2 interaction of the wild-type Sestrin protein but not of the Sestrin R407A mutant protein. d, Quantification of Sestrin protein associated with WDR24 protein in c. Flag–WDR24 was used as loading control. Sestrin protein levels were normalized to the sample without amino acid addition. n = 3 independent experiments. e, CRISPR–Cas9 strategy to generate the Sestrin R407A mutation. A Myc tag (orange) was inserted together with the R407A mutation (red). ATGPA and ATGPB indicate the start codons of the two Sestrin isoforms. fi, TORC1 activation on amino acid induction in fat bodies of third instar larvae. Fat bodies were starved for 1 h and then stimulated with leucine (f,g) or all amino acids (AA) (h,i) for 10 min. Lysates were analysed by immunoblotting. f,h, Representative immunoblots with leucine (f) and all amino acid (h) stimulation. g,i, Quantifications of blots with leucine (g) and all amino acid (i) stimulation.The Sestrin R407A mutation blocked TORC1 activation on induction with leucine (f,g) and all amino acids (h,i). n = 4 biologically independent sets of samples. Two-way analysis of variance (ANOVA) confirmed that SesnR407A mutants responded to addition of leucine and all amino acids significantly differently from wild-type fat bodies (P < 0.05). Data in d,g and i are presented as mean ± s.e.m. Statistics in d,g and i: two-way ANOVA with Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

To investigate how the Sestrin R407A mutation affected amino acid sensing by the TORC1 pathway in flies, we made amino acid starvation and stimulation experiments using the fat body of Drosophila larvae, because it responds rapidly to changes in nutrient availability, including amino acids37,38. When larval fat bodies were deprived of amino acids, phosphorylation of S6 kinase (S6K), a direct downstream target of TORC1, was low and did not differ between Sesnwt and SesnR407A mutants (Fig. 2f,g). We first measured the response of pre-starved larval fat body tissue to leucine stimulation, which resulted in an approximately threefold increase in pS6K levels in Sesnwt controls (Fig. 2f,g). In contrast, there was no increase in pS6K levels in fat bodies of SesnR407A mutants (Fig. 2f,g). The SesnR407A mutation therefore interfered with leucine-mediated activation of TORC1. We next asked whether the SesnR407A mutation would also block the activation of TORC1 signalling by all amino acids together. In Sesnwt flies, we observed a much greater approximately tenfold increase in pS6K levels on stimulation with all amino acids than with leucine treatment alone (Fig. 2h,i). One or more amino acids in addition to leucine are therefore required for full activation of TORC1 activity in the larval fat body. Interestingly, the increase in TORC1 activity in response to stimulation by all amino acids was almost completely blocked in SesnR407A mutants (Fig. 2h,i). This suggests that the SesnR407A mutation blocks not only leucine-mediated TORC1 stimulation but also stimulation by other amino acids, consistent with the finding that isoleucine, valine and methionine were also able to interrupt Sestrin–GATOR2 interaction (Fig. 2c,d). The result also indicates that the inhibitory effect of the amino acid-insensitive Sestrin R407A protein on TORC1 activity cannot be compensated by other amino acid-sensing mechanisms.

We next addressed whether the SesnR407A mutation also affected TORC1 activity under physiological conditions. Immunoblotting analysis on fat bodies of third instar larvae growing on normal fly food showed that pS6K levels were reduced by ~60% in SesnR407A mutants compared with Sesnwt controls (Fig. 3a,b), indicating chronically reduced activity of TORC1 in SesnR407A mutant larvae. We also tested whether the SesnR407A mutation affected activity of the TORC2 complex, by measuring phosphorylation of protein kinase B (AKT) at serine residue 505 (Ser505). Ser505 pAKT levels did not differ between SesnR407A mutant and Sesnwt controls (Fig. 3a,c), demonstrating that the SesnR407A mutation affected only the activity of the TORC1 complex. Finally, we measured threonine 184 phosphorylation of AMPKα, another downstream target of Sestrin, and found that it did not differ between SesnR407A mutant and Sesnwt control larvae (Extended Data Fig. 3a,b). The amino acid-sensing defective Sestrin R407A mutant protein therefore specifically downregulated TORC1 activity, and not activity of the TORC2 complex or of AMPK.

Fig. 3: The SesnR407A mutation impairs growth, increases lifespan and blunts high-protein-diet-induced lifespan shortening.
figure3

ac, SesnR407A mutant flies showed reduced TORC1 activity. Fat body tissues from third instar larvae grown under standard conditions were subjected to immunoblotting analysis (a). pS6K was normalized to total S6K (b), and pAKT to total AKT (c). pS6K, but not pAKT, was downregulated in SesnR407A mutant fat body tissues. n = 3 biologically independent samples. Data in b and c are presented as mean ± s.e.m. Unpaired, two-tailed t-test. d, SesnR407A mutant flies were developmentally delayed. Percentage eclosion at indicated time points. n = 10 vials with 50 embryos per vial. Data are presented as mean ± s.e.m. Permutation test showed that SesnR407A mutants emerged significantly later than wild-type flies. P value was adjusted for multiple testing. e, Wet weight of newly emerged flies. n = 20 pairs of flies. Median, 25th and 75th percentiles, and Tukey whiskers are indicated. Unpaired, two-tailed t-test. f,g, Survival (f) and median lifespan (g) of Sesnwt and SesnR407A females under DR. Lifespan of SesnR407A mutant flies was slightly extended under DR conditions with a median lifespan increase of 11%, 14% on the 0.5× and 1.0× DR food, respectively. On the 2.0× food under fully fed conditions, however, SesnR407A mutant females showed a strong increase in lifespan with an increase in median lifespan of 40%. P = 0.34 (0.1× food), P = 9.8 × 10−5 (0.5× food), P = 9.0 × 10−8 (1.0× food), P = 1.8 × 10−20 (2.0× food), log-rank test. SesnR407A flies responded to DR food significantly differently from control flies, P < 0.0001, CPH analysis. n = 100 flies for each condition of each genotype. The DR lifespan assay was performed once, and 1.0× condition was independently repeated.

Source data

To further characterize the physiological consequences of the Sestrin R407A mutation in vivo, we measured development time and female fecundity of SesnR407A mutants, because both traits depend on amino acid availability. SesnR407A mutant flies were fully viable but had a significantly delayed development (Fig. 3d) and reduced adult body weight (Fig. 3e) compared with Sesnwt controls. To test whether these growth effects were cell autonomous, we used the Flippase recombinase (FLP)/Flippase recognition target (FRT) system to generate mosaic clones in the larval fat body. Cells homozygous for the SesnR407A mutation were significantly smaller than wild-type cells (Extended Data Fig. 3c,d), demonstrating that the mutant Sestrin R407A protein reduced cell growth in a cell autonomous manner. SesnR407A mutant females also showed a 30% decreased cumulative egg production, mainly caused by reduced fecundity early in life (Extended Data Fig. 3e,f). Amino acid-insensitive SesnR407A mutants therefore had delayed development, and reduced growth and fecundity, consistent with chronically reduced TORC1 activity.

The amino acid-insensitive Sesn R407A mutation blunts the shortening of lifespan on a high-protein diet

To test the role of amino acid sensing by Sestrin in response to DR, we measured the lifespan of SesnR407A mutant flies under DR conditions. SesnR407A mutants showed only a small survival advantage on the 0.5× and 1.0× DR food, with a median lifespan increase of 11% and 14%, respectively. In contrast, median lifespan was increased by 40% on the high yeast diet (2.0× SYA), mainly due to a greater decrease in survival of control flies on this diet (Fig. 3f,g). CPH analysis revealed that the interaction between genotype and diet treatment was significant (P < 0.0001), confirming that SesnR407A flies responded significantly differently from Sesnwt flies to DR. The SesnR407A mutation hence increased lifespan under non-starvation conditions and attenuated the negative effect of high dietary yeast on lifespan, which is therefore partially mediated through amino acid sensing by Sestrin.

Sestrin function is important for maintenance of intestinal stem cells and gut health by DR

We next addressed in which tissue Sestrin function is required to modulate the response of lifespan to DR. The fly gut has recently been identified as a key tissue for the organismal response to DR20,39. Maintenance of gut homeostasis largely relies on stem cell-mediated renewal of absorptive enterocytes23,40 and preservation of stem cell activity has been associated with increased survival in flies23. As there are no transient-amplifying cells in the adult fly gut, the mitotic marker phospho-histone 3 (pH3) directly reflects the activity of stem cells21,22. We measured the number of pH3-positive cells in guts of Sesn3F6 loss-of-function mutants and wDah control females under fully fed (2.0×) and DR (1.0×) conditions. Consistent with previous studies20, DR decreased stem cell activity in the gut of control flies, with a reduction of ~50% in pH3-positive cells per gut (Fig. 4a,b). In contrast, DR did not decrease stem cell activity in the gut of Sesn3F6 mutant flies, with no difference from the number of pH3-positive cells in guts of wDah controls on 2.0× food (Fig. 4a,b). pH3 staining provides a snapshot of stem cell activity at a given time point. To measure gut cell turnover rates directly, we used the esgts FlipOut (F/O) system, which marks stem cells and all their progeny by heat shock-induced expression of a green fluorescent protein (GFP) protein. GFP-marked regions indicate newly generated cells, and their area, when compared with the corresponding total gut area, indicates gut cell turnover rates. In line with the results from pH3 staining, wDah control flies showed an ~40% decrease in gut turnover rates under DR conditions (Fig. 4c,d). In contrast, we found no difference in gut cell turnover rates when Sestrin was knocked down specifically in ISCs using RNA interference (Fig. 4c,d). Sestrin function in ISCs is hence essential for reduced cell turnover and stem cell maintenance under DR conditions.

Fig. 4: Sestrin is important for maintenance of ISCs in response to DR.
figure4

a,b, Gut stem cell activity of wDah and Sesn3F6 flies under DR. a, Representative images from 20-day-old flies. Actively dividing stem cells are indicated by arrows. b, Quantification of pH3-positive cells in Sesn3F6 females under DR. n = 25 guts (wDah, Sesn3F6, 2.0×; Sesn3F6, 1.0×), n = 24 guts (wDah, 1.0×). c,d, Gut turnover rates on Sestrin knockdown (KD). c, Representative images after 7 d induction. d, Quantification of gut turnover rates in Sestrin knockdown flies under DR. n = 14 guts (wDah, 2.0×, 1.0×), n = 16 guts (Sesn KD, 2.0×), n = 17 guts (Sesn KD, 1.0×). e,f, Gut stem cell activity of SesnR407A mutant flies under DR conditions. e, Representative images from 20-day-old flies. f, Quantification of pH3-positive cells in SesnR407A mutants under DR. n = 25 guts (Sesnwt, 2.0×, 1.0×), n = 24 guts (SesnR407A, 2.0×, 1.0×). g,h, Gut turnover rates in SesnR407A mutant flies under DR. g, Representative images after 7 d induction. h, Quantification of gut turnover rates in SesnR407A mutants under DR. n = 14 guts (Sesnwt, 2.0×; SesnR407A, 1.0×), n = 15 guts (Sesnwt, 1.0×; SesnR407A, 2.0×). i,j, Quantification of gut turnover rates in SesnR407A mutants on addition of all amino acids (i) and addition of methionine and BCAAs (MBC) (j). n = 13 guts (i), n = 16 guts (j). Scale bars, 50 μm. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (b,d,f,hj). Outliers are shown as open circles. Interaction between diet and genotype was significant in b,d,f and hj: two-way ANOVA, P = 0.02 (b), P = 0.01 (d), P = 0.02 (f), P = 0.01 (h), P < 0.0001 (i), P = 0.047 (j). Statistics in b,d,f and hj: two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

We next measured stem cell activity in the amino acid-insensitive SesnR407A mutant flies. Consistent with wDah control flies, Sesnwt control flies had high levels of intestinal pH3-positive cells under fully fed conditions and reduced levels under DR conditions (Fig. 4e,f). In contrast, SesnR407A mutants showed strongly decreased ISC activity under full feeding, and DR did not further reduce the number of pH3-positive cells (Fig. 4e,f). Consistently, gut cell turnover rates were also low in SesnR407A mutants on the 2.0× diet and were not further decreased by DR (Fig. 4g,h). To test whether dietary amino acid restriction was causal in reduced gut cell turnover under DR conditions, we added back amino acids to the 1.0× DR food and measured intestinal cell turnover rates. Addition of all amino acids to the 1.0× DR diet increased gut cell turnover rates of Sesnwt control flies to a similar level to that observed on the 2.0× fully fed diet (Fig. 4i). Importantly, gut cell turnover rates of SesnR407A mutant flies were not affected by addition of amino acids (Fig. 4i). Furthermore, addition of leucine, isoleucine, valine and methionine, the four amino acids involved in the regulation of Sestrin activity, to the DR medium was sufficient to increase gut turnover rates in Sesnwt controls but not in SesnR407A mutants (Fig. 4j). Sensing of these amino acids by Sestrin in ISCs or their progeny cells hence regulates stem cell activity in response to changes in dietary amino acids. Improved intestinal stem cell homeostasis is associated with a healthier gut20. Thus, to address the role of Sestrin in maintenance of gut health under DR, we measured gut pathology in old flies using a dysplasia assay14,20 and epithelial barrier function using a Smurf assay41. In control flies, DR reduced age-related gut dysplasia and the proportion of flies with a Smurf phenotype (Extended Data Fig. 4a–f). In contrast, dysplasia and incidence of Smurfs of Sesn3F6 loss-of-function mutant flies was not improved by DR, while SesnR407A mutant flies showed lower incidence of both phenotypes that could not be further reduced by the DR treatment (Extended Data Fig. 4a–f). Thus, these results illustrate that Sestrin is an important regulator of gut health in response to dietary protein restriction.

Sestrin acts in ISCs to maintain stem cell function and gut health

To identify the cell types in which Sestrin acts to maintain gut health, we employed cell type-specific overexpression of Sestrin. First, we measured ISC activity on ubiquitous overexpression of Sestrin using the inducible daGS GeneSwitch driver in young (day 10), middle-aged (day 30) and old (day 50) flies (Fig. 5a,b). Consistent with a previous report23, we observed an increase of pH3-positive cells during aging under non-induced conditions (Fig. 5b). Sestrin overexpression had only a small effect on stem cell activity in young flies, but prevented the age-related increase. Generalized linear modelling indicated that stem cell activity increased significantly less with age when Sestrin expression was induced (P < 0.05). Ubiquitous Sestrin overexpression was thus sufficient to preserve proliferative capacity of ISCs during aging. To determine which cell type underlay this effect, we measured pH3-positive cell numbers in middle-aged and old flies, where we observed the strongest effects. We used the 5966GS driver24 to overexpress Sestrin specifically in absorptive enterocytes, and did not observe reduced stem cell activity (Fig. 5c). In contrast, induction of Sestrin expression specifically in stem cells by using the 5961GS driver23 led to a clear reduction in stem cell activity (Fig. 5d). These results together imply that the function of Sestrin in ISCs, but not in enterocytes, is important for its role in stem cell maintenance.

Fig. 5: Sestrin overexpression in gut stem cells improves gut homeostasis.
figure5

a,b, Ubiquitous overexpression of Sestrin reduced gut stem cell activity. a, Representative images from 30-day-old flies without (RU486−) or with (RU486+) induction. Actively dividing stem cells are indicated by arrows. Scale bar, 50 μm. b, Quantification of pH3-positive cells on days 10, 30 and 50. n = 20 guts (RU486−), n = 22 guts (RU486+), day 10; n = 23 guts (RU486−, RU486+), day 30; n = 24 guts (RU486−, RU486+), day 50. Generalized linear modelling analysis confirmed that Sestrin overexpression significantly reduced the increase in age-dependent stem cell activity (P = 0.012). c, Overexpression of Sestrin in enterocytes (5966GS) did not affect pH3-positive cell numbers. Day 30: n = 22 guts (RU486−), n = 21 guts (RU486+); day 46: n = 22 guts (RU486−), n = 20 guts (RU486+). d, In contrast, overexpression of Sestrin in gut stem cells (5961GS) significantly reduced stem cell activity. Day 30: n = 24 guts (RU486−), n = 25 guts (RU486+); day 46: n = 22 guts (RU486−), n = 23 guts (RU486+). e, Sestrin overexpression (OE) prolonged the G1 phase in gut stem cells. Fly-Fucci and Sestrin were driven by the esg-Gal4 driver. Sestrin overexpression significantly changed the cell cycle distribution of gut stem cells. n = 416 cells, wDah; n = 382 cells, Sesn OE. Chi-square test. fh, Sestrin overexpression in gut stem cells decreased gut dysplasia (f,g) and the proportion of Smurf flies (h). f, Representative gut images from 50-day-old flies. DNA (DAPI) in blue. The gut epithelium is indicated by dashed lines. Scale bar, 20 μm. g, The difference in gut dysplasia was significant. n = 13 guts. h, Flies were 60 days old. n = 15 vials. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (bd,g,h). Outliers are shown as open circles. Statistics in bd: two-tailed, Mann–Whitney test; statistics in g and h: unpaired, two-tailed t-test.

Source data

To address how Sestrin affects stem cell function, we first measured their cell cycle state on Sestrin overexpression using the Fly-Fucci system, which relies on fluorochrome-tagged proteins to distinguish G1, S and G2 of the cell cycle. Sestrin overexpression in ISCs increased the proportion of cells in the G1 phase (Fig. 5e). In addition, a prolonged G1 phase indicates an inhibition of the G1–S transition, a phenotype known to be caused by reduced TORC1 activity42. To further test whether Sestrin function in ISCs was also causal for the improved gut health, we performed gut dysplasia and Smurf assays. We found that Sestrin overexpression reduced gut dysplasia (Fig. 5f,g) and improved gut barrier function (Fig. 5h). These results demonstrate that ISC-specific Sestrin expression improves gut homeostasis.

Sestrin regulates lifespan and intestinal stem cell maintenance via the TOR pathway

Sestrin has been identified as an amino acid sensor upstream of TORC133. To test whether the lifespan and stem cell phenotypes we observed in Sestrin mutants were indeed caused by regulation of the TOR pathway, we used the drug rapamycin to specifically inhibit TOR in Sestrin mutant flies. If Sestrin works exclusively via the TOR pathway, rapamycin treatment should revert the detrimental phenotypes caused by loss of Sestrin function and should not cause additive effects when combined with the amino acid-insensitive SesnR407A mutation. Consistent with this hypothesis, rapamycin treatment increased lifespan of Sesn3F6 mutant flies and, while Sesn3F6 mutant flies were short lived compared with controls in the absence of the drug, there was no difference in lifespan compared with wild-type control flies on the rapamycin diet (Fig. 6a). Furthermore, rapamycin treatment did not extend the lifespan of the already long-lived SesnR407A mutant flies (Fig. 6b), suggesting that Sestrin acts upstream of the TOR pathway to affect lifespan. We next investigated whether the same hypothesis also holds true for the ISC phenotypes. Rapamycin treatment reduced the increased ISC activity of Sesn3F6 loss-of-function mutant flies and there was no difference between wild-type controls and SesnR407A mutants on rapamycin treatment (Fig. 6c–f). Furthermore, rapamycin treatment blocked the increase in gut cell turnover upon knockdown of Sestrin in ISCs, and there were no additive effects on gut cell turnover rate when rapamycin was fed to flies overexpressing Sestrin in these cells (Fig. 6g–j and Extended Data Fig. 5a,b). Taken together, these results suggest that Sestrin regulates lifespan and ISC activity via the TOR pathway.

Fig. 6: Sestrin regulates lifespan and stem cell maintenance via the TOR pathway.
figure6

a,b, Survival of Sestrin mutant females under rapamycin treatment. a, Untreated Sesn3F6 flies were short lived (P = 3.7 × 10−8, log-rank test). Rapamycin treatment increased median lifespan of wDah and Sesn3F6 females by 13% and 21%, respectively. P = 6.4 × 10−11 (wDah), P = 2.1 × 10−29 (Sesn3F6), log-rank test. Lifespan of wDah and Sesn3F6 flies under rapamycin treatment was not significantly different (P = 0.30, log-rank test). Sesn3F6 flies responded to rapamycin treatment significantly differently from wDah, P < 0.001, CPH analysis. b, Lifespan of Sesnwt, but not of SesnR407A mutants, was significantly increased by rapamycin treatment. P = 1.0 × 10−6 (Sesnwt), P = 0.37 (SesnR407A), log-rank test. Lifespan of Sesnwt and SesnR407A flies under rapamycin treatment was not significantly different (P = 0.13, log-rank test). SesnR407A flies responded to rapamycin treatment significantly differently from Sesnwt, P < 0.001, CPH analysis. n = 150 flies. Experiments were performed once. cf, Gut stem cell activity of controls and Sesn mutant flies under rapamycin treatment. c,e, Representative gut images from 30-day-old flies: Sesn3F6 mutants flies (c) and SesnR407A mutant flies (e). Actively dividing stem cells are indicated by arrows. d,f, Quantification of pH3-positive cells in Sesn3F6 mutant flies (d) and in SesnR407A mutant flies (f) under rapamycin treatment. n = 21 guts (d) and n = 20 guts (f). Interaction between genotype and drug treatment was significant: two-way ANOVA, P = 0.0175 (d), P = 0.001 (f). gj, Gut cell turnover rates under rapamycin treatment on knockdown or overexpression of Sestrin. g,i, Representative images after 10 d of induction: Sestrin knockdown (g) and Sestrin overexpression (i). h,j, Quantification of gut cell turnover rates in Sestrin knockdown (h) and overexpression (j) flies under rapamycin treatment. n = 18 guts (h,j). The interaction between drug treatment and genotype was significant: two-way ANOVA, P < 0.0001 (h,j). Scale bars, 50 μm. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (d,f,h,j). Outliers are shown as open circles. Statistics in d,f,h and j: two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

Sestrin increases autophagy in ISCs to regulate gut cell turnover

Autophagy is a key downstream mediator of the TORC1 pathway43, and increased autophagy is essential for rapamycin-mediated longevity in Drosophila27. To investigate whether Sestrin regulates autophagy in ISCs, we used a fluorescence-tagged Atg8a reporter, UAS-mCherry::Atg8a, as a read-out for levels of autophagy. The esg-Gal4 driver was used to express this reporter in ISCs, and GFP was co-expressed to mark these cells. mCherry-positive cells were counted and the ratio to total GFP cells was calculated. A low basal level of autophagy was detected in wild-type stem cells. Interestingly, a significant increase of approximately threefold was detected when Sestrin was overexpressed (Fig. 7a,b). We next clarified whether the accumulation of the mCherry signal was indeed due to induced autophagy, or instead attributable to a block of autophagic flux, by using the UAS-GFP::mCherry::Atg8a reporter44,45. When this reporter was co-expressed with Sestrin, the dominant signal we detected was mCherry (Extended Data Fig. 5c), indicating that autophagic flux was not impaired. These results suggest that Sestrin induces autophagy in ISCs, probably by inhibiting TORC1 activity. We next addressed whether the increase in autophagy was functionally relevant for gut cell turnover. Therefore, we first overexpressed Atg1, a key upstream regulator of autophagy43, in ISCs and measured gut cell turnover rates. Overexpression of Atg1 reduced gut cell turnover rates (Fig. 7c,d), suggesting that induction of autophagy is sufficient to block gut turnover. Next, we blocked autophagy in ISCs by knocking down Atg5 via RNA interference46. Atg5 knockdown did not have an effect on gut cell turnover in the wild-type background, but it blocked the effect of Sestrin overexpression on gut cell turnover (Fig. 7c,d). Taken together these results identify increased autophagy in ISCs as an essential downstream mechanism by which Sestrin regulates gut physiology.

Fig. 7: Sestrin increases autophagy in ISCs to regulate gut cell turnover.
figure7

a,b, Sestrin overexpression induced autophagy in gut stem cells. Stem cells were marked by GFP (green), DNA by DAPI (blue) and autophagy by mCherry::Atg8a (gray). a, Representative images from 10-day-old females. b, Quantification of the proportion of mCherry::Atg8a-positive cells. Scale bars, 5 μm. n = 5 guts. Data are presented as mean ± s.e.m. Unpaired, two-tailed t-test. c,d, Autophagy is an essential downstream mediator of Sestrin function in ISCs. c, Representative images after 10 d of induction. Scale bar, 50 μm. d, Atg1 overexpression in ISCs reduced gut turnover rates and there was no additive effect when combined with Sestrin overexpression. Sestrin overexpression significantly reduced gut cell turnover rate, and this effect was blocked by Atg5 knockdown. n = 16 guts (wDah, Sesn OE, Atg1 OE, Sesn OE + Atg1 OE), n = 18 guts (Atg5 KD, Sesn OE + Atg5 KD). Gut cell turnover rates of Sestrin overexpression flies showed a significantly different response to Atg1 overexpression (two-way ANOVA, P = 0.0001) and Atg5 knockdown (two-way ANOVA, P = 0.0023) than that of wild-type flies. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (d). Outliers are shown as open circles. Statistics in d: two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

Sestrin overexpression in ISCs extends lifespan

Improved ISC maintenance has been associated with increased organismal survival in flies23. Thus, we next addressed whether ISC-specific overexpression of Sestrin would be sufficient to increase adult survival. We first measured survival of adult flies in which Sestrin was ubiquitously overexpressed under the control of the daGS driver, which significantly increased lifespan, with a median extension of 10% (Fig. 8a). Overexpression of Sestrin in neurons or fat bodies using the elavGS and S1106GS drivers, respectively, did not cause lifespan extension (Fig. 8b,c), nor did overexpression using the enterocyte specific 5966GS driver (Fig. 8d). In contrast, overexpression of Sestrin under the control of the ISC-specific 5961GS driver resulted in a significant lifespan extension, again with a median lifespan extension of ~10% (Fig. 8e). Relative lifespan extension was even greater on a high yeast diet (2.0× SYA), with a 15% increase in median lifespan (Fig. 8f), suggesting that Sestrin function in stem cells protects against the lifespan-shortening effects of a protein-rich diet. Thus, Sestrin overexpression in ISCs could extend fly lifespan to the same extent as ubiquitous overexpression, suggesting that ISCs are a key cell type mediating the effects of Sestrin gain of function on survival.

Fig. 8: Sestrin overexpression in gut stem cells extends lifespan.
figure8

a, Median lifespan of female flies with adult-onset ubiquitous overexpression of Sestrin using the daGS driver was increased by 10% compared with the non-induced control (P = 2.2 × 10−5, log-rank test). bd, Adult-onset overexpression of Sestrin in the adult nervous system (elavGS) (b) (P = 0.82, log-rank test), the adult fat body (S1106GS) (c) (P = 0.15, log-rank test) or gut enterocytes (5966GS) (d) (P = 0.09, log-rank test) did not affect lifespan. e, Overexpression of Sestrin in gut stem cells (5961GS) significantly increased lifespan, with a median lifespan increase of 10% (P = 4.2 × 10−4, log-rank test). Lifespans in ae were performed on 1.0× SYA. f, Overexpression of Sestrin in gut stem cells increased median lifespan by 15% on a high-yeast diet (2.0× SYA) (P = 5.4 × 10−6, log-rank test). n = 100 flies (ae), n = 120 flies (f). The experiment in a was repeated three times, in e twice and in the others once.

Source data

In summary, our study identified the evolutionarily conserved Sestrin protein as a key regulator of stem cell activity under DR in Drosophila. Sestrin regulates TORC1 signalling and proliferation of ISCs in response to the availability of dietary amino acids. Therefore, our data establish Sestrin as a novel molecular link that mediates the effects of dietary amino acid restriction on ISC function and health and on organismal lifespan.

Discussion

Dietary restriction is so far the most effective intervention to lengthen life and healthspan in animals and short-term dietary interventions can also improve health in humans47. However, in humans, long-term compliance greatly limits its applicability to the wider population. Understanding the molecular mechanisms underlying the beneficial effects of DR could lead to development of novel treatments based on DR mimetics. Here we identified the evolutionarily conserved amino acid sensor protein Sestrin as a key regulator of stem cell function, intestinal health and survival in response to dietary amino acids. Thus, our findings implicate Sestrin as a novel target for anti-aging interventions.

Sestrin has been identified as an amino acid-sensing protein that acts as a negative regulator of TORC1 activity via the GATOR/Rag complex33,34. This role of Sestrin has mainly been studied in the context of acute amino acid stimulation of previously amino acid-starved cultured cells, and the in vivo relevance of Sestrin in amino acid sensing is still under debate35. Although it binds to several essential amino acids in vitro, in mammals Sestrin has been postulated to act mainly as a leucine sensor33,34. We found that leucine addition only resulted in a threefold increase in pS6K levels, a proxy for TORC1 activity, while addition of all amino acids induced a tenfold increase in larval fat bodies. This result is notable given that leucine was supplied at a 20-fold higher concentration compared with the addition of all amino acids. Thus, a combination of several amino acids is needed for full activation of TORC1 signalling in the Drosophila fat body. Importantly, leucine-induced TORC1 activation was completely dependent on Sestrin, implying that there are no other mechanisms leading to leucine sensing by TORC1. TORC1 activation by all amino acids was also strongly reduced in amino acid-insensitive SesnR407A mutants, confirming that other amino acids in addition to leucine are required for full activation of TORC1 activity by Sestrin. In summary, our results indicate that in vivo Sestrin senses not only leucine but also the other BCAAs and methionine.

Sestrin has been previously studied in both flies36 and mice48,49,50, although not in the context of dietary amino acid sensing. Sestrin mutant flies exhibit a broad range of age-associated pathologies36 but, surprisingly, were not short lived31. Our findings indicate that longevity of Sestrin mutants is dependent on the nutritional context, with mutant flies short lived compared with controls specifically under DR conditions, but not on a high-protein diet. This is consistent with the chronically increased activity of the TORC1 pathway in Sestrin mutant flies36, which is expected to have a bigger detrimental effect under DR conditions than on a high-protein diet, where TOR activity is already high.

Reduced TOR signalling is an important downstream mediator of DR benefits27,51. Lowered dietary amino acids have been shown to be causal for DR-induced longevity in flies11, but the molecular mechanisms mediating these benefits and the tissues they act in are still elusive. Our results establish Sestrin as an important mediator of DR benefits; however, loss of Sestrin did not fully block the beneficial effects of DR on longevity and the combination of DR and the SesnR407A mutation resulted in a greater extension of lifespan than each treatment individually, indicating that DR and TOR might also work via independent mechanisms. This hypothesis is consistent with recent gene expression studies in flies and mammals, which suggest both shared and independent mechanisms of DR and reduced TOR signalling52,53,54.

The gut is a key tissue mediating health benefits of DR and reduced TOR signalling20,25,55,56. Importantly, we showed that restriction of dietary amino acids is causal for improved gut homeostasis under DR. Consistent with the binding affinity of Sestrin in vitro, BCAAs and methionine seem to play a causal role in this context. Concordantly, restriction of BCAAs has recently been shown to be sufficient to improve gut homeostasis in flies14. Noteworthy, also restriction of three non-BCAAs caused improved gut homeostasis14, which suggests the involvement of additional amino acid-sensitive mechanisms in addition to Sestrin. The tuberous sclerosis complex 2 (TSC2) protein is implicated in amino acid-dependent regulation of TORC157, and the TSC1/2 complex regulates ISC maintenance in flies via the TORC1 pathway58,59,60. Thus, Sestrin and TSC2 might act in concert or in parallel in regulating amino acid-dependent regulation of stem cell maintenance. Interestingly, the SesnR407A mutation blocked gut turnover rates also on supplementation of all amino acids, which suggests that strong Sestrin gain of function in ISCs can override the input of other amino acid-sensing systems.

DR and the TOR pathway also play important roles in maintenance of adult stem cells in mammals61,62,63. The effect of DR on stem cell maintenance can be indirect, via TORC1 signalling in stem cell niche cells, or via regulation of TORC1 directly in stem cells. For example, DR caused downregulation of TORC1 activity in the radioresistant, reserve ISC pool, which contributes to enhanced tissue regeneration after DR64. Importantly, leucine supplementation was sufficient to induce TORC1 activity in reserve ISCs64, implicating amino acid-sensing systems in TORC1-mediated regulation of ISC maintenance in mammals. However, whether the mammalian Sestrin homologues are involved in this regulation is currently unknown.

We show that Sestrin regulates lifespan via the TOR pathway and established autophagy as an essential mediator downstream of TOR that regulates gut cell turnover. Autophagy is required for stem cell maintenance and intestinal homeostasis in flies65 and mammals66,67; however, whether upregulation of autophagy in ISCs is sufficient to extend lifespan is currently unknown. Additional mechanisms downstream of TORC1, such as inhibition of S6K27 and of RNA Pol III68 have also been implicated in ISC maintenance and longevity, and therefore might also contribute to the effect of Sestrin in ISCs. In summary, our results establish Sestrin as a novel molecular link that mediates the effects of dietary amino acid restriction on TORC1 activity in stem cells of the fly gut, thereby maintaining gut health and ensuring longevity.

Methods

Fly stocks and husbandry

All fly stocks were backcrossed for at least six generations into the wDah background, with the exception of tool chromosomes in the clonal assay69, the gut turnover assay70 and the Fly-Fucci system71. Flies were maintained on 1.0× SYA (10% (w/v) brewer’s yeast, 5% (w/v) sucrose and 1.5% (w/v) agar) food at 25 °C, 60% humidity, on a 12 h light and 12 h dark cycle condition, unless otherwise noted. To induce gene expression using the GeneSwitch system72, RU486 (Sigma, catalogue number M8046) was added to the food at a final concentration of 200 μM. Rapamycin (LC Laboratories, catalogue number R-5000) was added to the food at a final concentration of 200 μM. Female flies were used unless otherwise noted. The fly stocks used in this study are listed in Supplementary Table 1, and the genotypes of flies in each figure are listed in Supplementary Table 2.

Sequence alignment and structure modelling

Sestrin protein sequence alignment was performed using the PROMALS3D method73. Sequences of each species used in the analysis were as follows: UniProt: Q5W1K5 (Drosophila melanogaster), UniProt: P58004 (Homo sapiens), UniProt: P58043 (Mus musculus), UniProt: Q6NU55 (Xenopus laevis), UniProt: A0F081 (Danio rerio), UniProt: Q9N4D6 (Caenorhabditis elegans). Sestrin protein structure was modelled using UCSF Chimera V1.12.074. Human Sestrin2 protein structure (PDB 5DJ4) was used as a modelling template.

Generation of mutant and transgenic fly lines

To generate the transgenic UAS-Sestrin fly line, the full-length Sestrin complementary DNAs were amplified by PCR from BDGP DGC clone (LD39604) and cloned into pUAST–attB vector75. φC31-mediated transgenesis75 was used to generate transgenic flies, using the attP2 insertion site76. The Sesnwt and SesnR407A fly lines were generated by a fully transgenic CRISPR–Cas9 approach using two guide RNAs (gRNAs)77 targeting the Sestrin gene locus. The gRNAs were amplified by PCR using the pCFD4 plasmid as template, followed by its cloning into the pCFD4 vector using the Gibson Assembly kit (NEB, catalogue number E2611S). The resulting plasmid was then introduced to the attP40 insertion site76 to generate Sestrin–gRNA transgenic flies. The Sestrin donor constructs contained the following elements: left homologous arm (1,045 bp); PAM site (mutated to NheI); R407 region (3,718 bp); tag region (679 bp); PAM site (mutated to KpnI); right homologous arm (1,015 bp). These fragments were amplified from BAC clone (BACR02E09) and cloned into pOT2 vector78. The Myc tag was introduced using sequences in primers, and the Sestrin R407A mutation was generated by site-directed mutagenesis (Agilent). To generate Sesnwt and SesnR407A mutant flies, flies expressing the two gRNAs were crossed with flies expressing Cas9 under the ubiquitous actin promotor, and their progeny embryos were injected with the Sesnwt::myc and SesnR407A::myc donor constructs. PCR screening targeting the Myc tag sequence was used to identify positive lines and the presence of the R407A mutation was confirmed by sequencing. Primers used for cloning are listed in Supplementary Table 3.

Development time and adult weight

For development time analysis, flies were allowed to lay eggs for 60 min, and these were transferred to vials at a density of 40 or 50 per vial, as indicated in each experiment. The number of flies eclosed was then recorded. For body weight measurements of adult flies, 40 flies that eclosed within 12 h were anaesthetised and weighted in pairs on an ME235S genius balance (Sartorius).

Lifespan, dietary restriction and fecundity

For lifespan assays, flies were reared at standard larval density (20 μl embryos = 300 eclosed flies, per bottle). Eclosed flies were allowed to mate for two days, and then sorted into vials. Flies were transferred to fresh vials every two to three days and scored for deaths. In most experiments, 1.0× SYA food was used. For DR experiments, the yeast content of the diet was diluted with 2.0× SYA containing 200 g yeast per litre of food, 1.0× SYA containing 100 g yeast per litre of food, 0.5× SYA containing 50 g yeast per litre of food or 0.1× SYA containing 10 g yeast per litre of food26. Details of all lifespan trials are listed in Supplementary Tables 4 and 5. For the fecundity assay, eggs laid in a period of 3–6 h within the first 4 weeks of adulthood were collected and counted, twice a week in the first two weeks and once a week in the next two weeks.

RT–qPCR

Total RNA was extracted from flies using Trizol (Invitrogen, catalogue number 15596018) following the manual. RNA concentration was measured by Nanodrop One (Thermo Scientific). cDNA synthesis was performed using the SuperScript ΙΙΙ first-strand synthesis kit (Invitrogen, catalogue number 18080-400) with random hexamers and 600 ng total RNA as input. SYBR Green Master Mix (Applied Biosystems, catalogue number 4367659) was used and samples were prepared with the Janus automated workstation (PerkinElmer). qPCR was performed using the QuantStudio 6 Flex real-time PCR system with Real-Time PCR Software V1.1 (Life Technologies). Relative expression levels were determined using the ΔΔCT method, and Rpl32 was used for normalization. Primers for RT–qPCR are listed in Supplementary Table 3.

Sestrin–GATOR2 interaction assay

HEK-293T cells (ATCC, catalogue number CRL-3216) were cultured in Dulbecco’s modified Eagle medium (Gibco, catalogue number 41965039) supplemented with 10% fetal bovine serum (Gibco, catalogue number 10270-106), 50 units per ml penicillin and 50 μg ml−1 streptomycin (Gibco, catalogue number 15070-063). Cells were maintained at 37 °C in a 5% CO2 incubator (Thermo Scientific). HEK-293T cells were plated in 10 cm plates one day before transfection. Cell transfection was performed using polyethylenimine (Sigma, catalogue number 408727) with pcDNA3-based plasmids expressing fly cDNAs (WDR24 cDNA source: BDGP DGC clone LD21720) for Flag–WDR24 and HA–Sestrinwt or HA–SestrinR407A. The transfected amounts for each plate were as follows: 5 μg DNA per vector (total 10 μg) was used for transfection; 48 h after transfection, cells were starved of all amino acids for 50 min and then collected. Cells were lysed with Triton lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM MgCl2, and 1× Complete protease inhibitors (Roche, catalogue number 11836170001) and 1× PhosSTOP phosphatase inhibitors (Roche, catalogue number 04906837001). Cell lysates from the same type of transfected cell plates were combined, mixed and then equally distributed to test tubes. Individual amino acids were added to the cell lysate at a final concentration of 2 mM, a concentration within the physiological range for most essential amino acids in Drosophila cells (Supplementary Table 6). Anti-Flag microbeads were added to cell lysate, followed by incubation with rotation for 2 h at room temperature. Anti-Flag immunoprecipitation was performed according to the manual of the μMACS Flag Isolation Kit (Miltenyi Biotec, catalogue number 130-101-591) with the wash buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 8.0), 500 mM NaCl and 2.5 mM MgCl2. Immunoprecipitated proteins were analysed by immunoblotting using antibodies against Flag and HA tag.

Amino acid concentration measurement in cultured cells

Drosophila S2R+ cells (DGRC, catalogue number 150, CVCL_Z831) were cultured in Schneider’s Drosophila medium (Thermo Fisher Scientific, catalogue number 21720024) supplemented with 10% fetal bovine serum (Gibco, catalogue number 10270-106), 50 units per ml penicillin and 50 μg ml−1 streptomycin (Gibco, catalogue number 15070-063). Cells were maintained at 25 °C in an incubator (Thermo Scientific). Cells were incubated in fresh medium for 2 h and collected. Number and diameter of cells were measured by Vi-CELL XR cell viability analyser with software 2.04 (Beckman Coulter). The collected cell pellets were subjected to LC-MS analysis. Identity of each compound was validated by authentic reference compounds. Data analysis of the measured amino acids was performed using the TraceFinder software (V4.2, Thermo Fisher Scientific). Amino acid concentration was calculated based on the amount of amino acids and the cell numbers and volumes of samples.

Amino acid starvation and stimulation

Amino acid-free or -containing media were made based on the formula of Schneider’s Drosophila medium (1×, Thermo Fisher Scientific, catalogue number 21720024). Stock solutions were made and mixed to get a final 1× medium. Fat bodies were dissected in Schneider’s Drosophila medium within 30 min. Samples were rinsed twice with amino acid-free medium and then incubated for 1 h in the same medium. Amino acid stimulation was performed by addition of medium containing only leucine (final concentration, 3 g l−1, 20×) or by addition of medium containing all amino acids (final concentration, 1×) for 10 min. Samples were immediately lysed in Laemmli buffer and subjected to immunoblotting analysis.

Immunoblotting

Tissues were lysed in the RIPA buffer or the Laemmli buffer with addition of 2× Complete protease inhibitors (Roche, catalogue number 11836170001) and 2× PhosSTOP phosphatase inhibitors (Roche, catalogue number 04906837001). Protein extracts were resolved using SDS–PAGE and transferred to Immobilon-FL PVDF membranes (Merck, catalogue number IPFL00010). Blots were blocked in Odyssey blocking buffer (LI-COR, catalogue number 927-50000) for 1 h and probed with the following antibodies: anti-Sestrin (1:1,000), anti-Myc (1:1,000), anti-Flag (1:1,000), anti-HA (1:1,000), anti-pS6K T398 (1:1,000), anti-S6K (1:1,000), anti-tubulin (1:5,000), anti-pAMPKα T172 (1:1,000), anti-pAKT S505 (1:1,000), anti-AKT (1:1,000), goat anti-rabbit IgG IRDye 680RD (1:15,000), goat anti-mouse IgG IRDye 800CW (1:15,000). Detection and quantification were performed using the Odyssey Infrared Imaging system with application software V3.0.30 (LI-COR). The sources of antibodies used are listed in Supplementary Table 7.

Immunofluorescence

Tissues were dissected in PBS and collected on ice. Samples were fixed 30 min with 4% formaldehyde (Thermo Scientific, catalogue number 28908) and blocked with 5% non-fat milk for at least 2 h at room temperature. Primary antibody incubation was at 4 °C overnight, followed by secondary antibody incubation for at least 2 h at room temperature. The antibodies used were the following: anti-pH3 (1:200), goat anti-rabbit IgG, AF594 (1:1,000). The sources of antibodies used are listed in Supplementary Table 7. Samples were mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, catalogue number H-1200). Imaging was performed using Leica TCS SP5-X, SP8-X confocal microscopes.

FLP/FRT clonal assay and gut turnover assay

For fat body FLP/FRT clonal assay69, eggs laid overnight were heat-shocked at 37 °C for 1 h. Fat bodies from wandering third instar larvae were dissected and fixed in 4% formaldehyde (Thermo Scientific, catalogue number 28908). Samples were stained with Phalloidin CF594 (1:40, Biotium, catalogue number 00045) for 30 min and mounted. For gut turnover assay, the esgts F/O flies (w; esg-Gal4, tubGal80ts, UAS-GFP; UAS-flp, Act>CD2>Gal4) were used70. Crosses were set up and progeny were raised at 18 °C. Four-day-old females were sorted and shifted to 29 °C for indicated days. For amino acid adding back to 1.0× SYA diet, the amount of amino acids was based on a previous study11. Guts were dissected, fixed in 4% formaldehyde (Thermo Scientific, catalogue number 28908) and mounted. Samples were imaged using Leica TCS SP5-X, SP8-X confocal microscopes with focus on R4-R5 region of the gut, and images were analysed using ImageJ 1.51m9.

Gut dysplasia and Smurf assay

Flies were maintained at indicated conditions and aged until the day of assays. For gut dysplasia assay, guts were dissected, fixed for 30 min with 4% formaldehyde (Thermo Scientific, catalogue number 28908), and then mounted to slides with Vectashield mounting medium containing DAPI (Vector Laboratories, catalogue number H-1200). Samples were imaged using Leica TCS SP8-X confocal microscopes with focus on the R2 region of the gut. Images were analysed using ImageJ. Proportion of dysplasia was scored as the ratio between length of gut epithelium with several layers of nuclei and total length of the gut. For Smurf assay, flies were transferred to standard SYA food containing 1.2% (w/v) blue dye (Sigma, catalogue number 861146) for 48 h. If the dye leaked outside of the gut, it was scored as Smurf.

Statistics and reproducibility

No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications20,23,68. Samples were allocated to groups/treatments randomly, and steps were taken to avoid batch effects. Experimental conditions were not blinded. However, data analysis was performed blind whenever possible. No data were excluded from the analysis. The times of experiments independently repeated were indicated in relevant figure legends for lifespan assays, and for other assays experiments were performed once with indicated setups. Data were analysed using the GraphPad Prism 6, JMP 10, R 3.3.1 or Excel 2013 software. For development time assays, data were analysed using permutation test (R, statmod package). For lifespan assays, data were recorded in Excel, and a log-rank test was performed. CPH analysis was performed using JMP software (SAS). Data presented Fig. 5b–d and in Extended Data Fig. 3e,f were not normally distributed, thus a Mann–Whitney test was used for the analysis. Other data distribution was assumed to be normal but this was not formally tested. For small sample sizes (N < 10), data are presented with mean ± s.e.m. with individual data point shown. For samples with large sizes (N ≥ 10), box-and-whisker plots are used with median, 25th and 75th percentiles, and Tukey whiskers indicated in plots. Outliers are shown as open circles. P values less than 0.05 are considered to be statistically significant, and exact P values are provided whenever possible in figures or figure legends. Additional details, including F values, t values and degrees of freedom for relevant tests can be found in the statistical Source Data files.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The structure of human Sestrin2 (Uniprot: P58004) used to model the structure of the fly Sestrin protein (Uniprot: Q9W1K5) is PDB 5DJ4 (https://doi.org/10.2210/pdb5DJ4/pdb). All data that support the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank J. H. Lee, B. Edgar, G. Juhasz, the Bloomington Stock Center and the VDRC Stock Center for fly strains and reagents. We are also grateful to all members of the Partridge Lab for helpful insights, and to C. Demetriades for critical comments. Imaging was performed in the FACS and Imaging Core Facility, and amino acid concentrations were determined in the Metabolomics Core Facility at the Max Planck Institute for Biology of Ageing. The work was supported by a Swiss National Science Foundation (SNSF) postdoc fellowship (P2BEP3_162093) to J.L. and by funding from the Max Planck Society to L.P. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 268739.

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J.L., S.G. and L.P. conceived and designed the study. J.L. conducted most experiments, U.T. and A.M.-H. provided assistance. J.E. contributed to the generation of transgenic flies. J.L. and S.G. analysed the data. J.L., S.G. and L.P. wrote the manuscript.

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Correspondence to Sebastian Grönke or Linda Partridge.

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Extended Data

Extended Data Fig. 1 Characterization of a Sestrin loss-of-function allele and its effects on fly development.

a, Genomic locus of the Sestrin gene. In Sesn3F6 mutant flies the first two non-coding exons of the Sestrin gene are deleted. b, qRT-PCR analysis showed strongly reduced Sestrin mRNA levels in Sesn3F6 mutant flies. Data are presented as mean ± SEM. N = 3 biologically independent samples. Unpaired two-tailed t-test. c, No Sestrin protein was detected in Sesn3F6 mutant flies by immunoblotting analysis. An additional control sample (Sesn8A11 null mutant) was loaded in between. d, Sesn3F6 mutant flies showed a decreased developmental time compared with wDah wild type control flies (N = 10 vials, 40 embryos each). Permutation test (R, statmod package) showed that Sesn3F6 mutants eclosed significantly earlier than wild type flies. P value was adjusted for multiple testing. e, Sesn3F6 mutant flies had increased body weight. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots. N = 20 pairs of flies, unpaired two-tailed t-test.

Source data

Extended Data Fig. 2 Sestrin mRNA and protein levels are not affected by the SesnR407A mutation.

a-c, qRT-PCR (a) and immunoblotting (b,c) analyses confirmed that there was no change in Sestrin mRNA or protein levels in SesnR407A mutant flies compared with Sesnwt control flies. The Myc tag was used to detect Sestrin proteins in immunoblotting. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test.

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Extended Data Fig. 3 SesnR407A mutation reduces cell growth and decreases fecundity.

a,b, SesnR407A mutation did not regulate pAMPKα activity. Fat body tissues from 3rd instar larvae grown under standard conditions were subjected to immunoblotting analysis. pAMPKα was normalized to Tubulin. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test. c,d, SesnR407A-dependent reduction of cell size was cell autonomous. Confocal microscopy images of SesnR407A mutant clones in larval fat bodies (c). DNA (DAPI, blue), Actin (Phalloidin, red), and GFP (green). GFP-negative cells, outlined with white dashed lines, indicate Sestrin homozygous clones. Size of homozygous cells was normalized to wild type twin clones (marked by bright GFP). Scale bar: 25 μm. (d) Quantification of cell size from confocal images. N = 13 (Sesnwt) and N = 14 (SesnR407A) independent fat body samples, unpaired two-tailed t-test. e, SesnR407A mutant flies showed reduced cumulative egg laying. N = 10 vials with 20 flies each, two-tailed, Mann–Whitney test. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (d, e). Outliers are shown as open circles. f, Reduced fecundity was restricted to early-life fecundity. Data are presented as mean ± SEM. N = 10 vials, two-tailed, Mann–Whitney test.

Source data

Extended Data Fig. 4 Sestrin is required for better maintenance of gut homeostasis by DR.

a-d, Sestrin function was required for preventing gut dysplasia from a high-protein diet. Gut dysplasia of wild type and Sesn mutant flies on fully fed (2.0x) and DR (1.0x) food was measured. (a,c) Representative gut images from 45 days old flies. DNA (DAPI) was blue. Epithelial layers are indicated by dashed lines. Scale bar represents 20 μm. (b, d) Quantification of gut dysplasia in Sesn3F6 (b) and in SesnR407A (d) mutant females under DR. N = 13 guts (b), N = 11 guts (d). Interaction between diet and genotypes was significant: two-way ANOVA, P = 0.024 (b), P = 0.011 (d). e,f, Sestrin was also required for maintenance of gut epithelial barrier function under DR. Smurf phenotypes of wild type and Sesn mutant flies on fully fed (2.0x) and DR (1.0x) food were scored at the age of 50 days. Proportion of Smurf flies in Sesn3F6 (e) and in SesnR407A (f) mutant females. N = 15 vials (e, f). Interaction between diet and genotypes was significant: two-way ANOVA, P = 0.04 (e), P = 0.02 (f). Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots ((b, d-f)). Outliers are shown as open circles. Statistics in (b,d-f): two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

Extended Data Fig. 5 Sestrin over-expression induces autophagic flux in gut stem cells.

a,b, Sestrin mRNA expression levels in RNAi-mediated knockdown and over-expression conditions. The ubiquitous, constitutive da-Gal4 driver was used to drive expression of each construct. (a) RNAi-mediated Sestrin knockdown significantly reduced Sestrin mRNA level, whereas (b) Sestrin over-expression significantly increased Sestrin mRNA level. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test. c, Autophagic flux in ISCs upon Sestrin over-expression. Sestrin and a GFP::mCherry::Atg8a reporter were co-expressed in ISCs using the esg-Gal4 driver. GFP in green, mCherry in red, and DAPI (DNA) in blue. A representative gut image from 6 guts was shown. A strong mCherry signal was detected when Sestrin was over-expressed. The lower panel shows the magnification of the inset in the upper panel. Scale bars: 20 μm.

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Lu, J., Temp, U., Müller-Hartmann, A. et al. Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids. Nat Aging 1, 60–72 (2021). https://doi.org/10.1038/s43587-020-00001-7

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