Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila

Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine¹. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation^2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn^-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (Sesn^L431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.

Extended Data Fig. 1:. Validation of chemically-defined diets and loss of Sestrin phenotypes in larval fat bodies.

a, b, Drosophila larvae eating chemically-defined diets lacking individual amino acids have reduced levels of the missing amino acid. Relative levels of leucine (a) and valine (b) measured by LC-MS/MS in whole larval extracts of Wild-type (OreR) or Sesn^L431E larvae fed the indicated diet for 4.5 hours. Values are mean ± SD of technical replicates from a representative experiment. n=4 independent biological samples. Two samples from wild type (OreR) leucine-free and valine-free, respectively, failed to yield decent peaks for leucine levels, thus discarded. Multiple unpaired t tests, Holm-Šídák multiple comparison method. c, Sesn knockdown prevents autophagy induction upon leucine deprivation. Fat body cells in mid-third instar larvae expressing mCherry-Atg8a were fed the indicated diets for 4.5 hours. The Sesn RNAi was expressed in clones of cells (GFP, outlined) with a FLP-out system. Scale bar, 10 μm. d, Loss of Sestrin does not affect the inhibition of mTORC1 caused by the deprivation of all food. Immunoblot analyses of phospho-S6K and S6K in adult female flies in the fed state or starved of all food for 1 day.

Extended Data Fig. 2:. The Sesn^L431E mutation does not affect adult fly lifespan on the chemically defined diets but does mildly delay larvae development, while loss of Sestrin does not affect larvae development.

a, Loss of Sestrin does not the affect development of larva feeding on a complete diet. Time to pupariation for w¹¹¹⁸ and Sesn ^−/− larvae fed the standard yeast-based diet. b-g, Survival curved for animals of the indicated sex and genotypes fed the indicated chemically-defined diets. (a) nWT(OreR)=235; n(Sesn^L431E)=238; (b) nWT(OreR)=233; n(Sesn^L431E)=237; (c) nWT(OreR)=242; n(Sesn^L431E)=248; (d) nWT(OreR)=242; n(Sesn^L431E)=240; (e) nWT(OreR)=229; n(Sesn^L431E)=243; (f) nWT(OreR)=245; n(Sesn^L431E)=245. See statistics in Supplementary Data 1 and methods. h. Sesn^L431E larvae raised on a standard yeast-based diet are developmentally delayed. Data are representative of three independent experiments with similar results. Statistical analysis was performed using a permutation test on the difference of the mean pupariation times of the two genotypes (a, h).

Extended Data Fig. 3:. Sestrin-mediated mTORC1 signaling in ovaries.

a, b, Sestrin mediates leucine-sensing by mTORC1 in adult animals. Immunoblot analyses of whole adult animals of the indicated sex and genotype following overnight starvation and 1.5 hours of refeeding with the indicated diets. c, In flies feeding a standard diet and lacking Sestrin or expressing the leucine-binding deficient Sestrin mutant (L431E), mTORC1 activity is increased or decreased, respectively. Lysates were prepared from isolated ovaries from animals of the indicated genotypes and fed a standard yeast-based diet. d, e, Loss of Sestrin accelerates the reduction in ovary size caused by leucine starvation. (d) Ovarian size in females of the indicated genotypes fed the indicated diets for 24 hours. Results are quantified in (e). Scale bar, 500 μm. f, g, Sesn^L431E flies have reduced fecundity but not fertility. (f) Number of eggs laid over a period of 60 hours by females of the indicated genotypes maintained on the standard yeast-based diet. (g) Hatching rate of eggs laid in the same conditions as in (f). (e, f, g) Values are mean ± SD of technical replicates from a representative experiment. (e) n=6 (Wild type (w¹¹¹⁸)), 8 (Sesn ^−/−), 7 (Sesn^L431E amino acid-replete diet), 5 (Sesn^L431E leucine-free diet), and 6 (Sesn^L431E valine-free diet). (f) n=5. (g) n=4. Data are representative of three independent experiments with similar results. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test (e), and one-way ANOVA (f, g) followed by Dunnett’s multiple comparisons test.

Extended Data Fig. 4:. Sestrin mediates the preference for leucine-containing food and influences total food intake.

a-c, Characterization of the methods used in the food two-choice assay. (a) Measurement of the weight of the apple pieces used in the assay. n=8. (b) Background qPCR signal determination for each oligonucleotide barcode used in assay. n=6 for each condition. (c) The qPCR signals used to determine the leucine preference of the wild-type flies come primarily from internal DNA oligonucleotides instead of external ones that might contaminate the outside of the body of female flies. qPCR for oligonucleotide barcodes in a leucine versus water choice assay before and after washing animals as previously described. n=4 for both pre and post wash conditions. d, Preference of the flies for apple pieces painted with the indicated leucine concentrations. Animals were given a choice between leucine- or water-coated apples. Indicated leucine concentrations (5 mM, 15 mM, 30 mM, and 70 mM) were the solution concentrations used to coat apples. The final concentration on the food should be ~10 times more diluted. n (5 mM) = 7, n (15 mM and 30 mM) = 6, n (70 mM) = 5. e, Adult female flies do not have a preference for valine- versus water-painted apple pieces. Wild-type (OreR) animals were given indicated food choices and the preference fold-difference was shown. n (leucine vs water) = 8, n (valine vs water) = 10, n (leucine vs valine) = 7. f, Rapamycin treatment reduces fly food consumption. Vehicle or Rapamycin pre-treated animals were given a choice between leucine- or water-coated apples. For the Rapamycin group during the choice assay, animals were fed on apples painted with Rapamycin in addition to either leucine or water. Data show the normalized values of food consumption. n=5 for both conditions. g, Sesn^L431E animals do not have a preference for valine- over water-painted apple. Animals were given a choice between valine- or water-coated apples and food preference was measured at the indicated time points. Data show the fold-difference in relative food intake for the valine-coated apple compared to the water-coated apple. n=10 (2 hrs), 12 (4 hrs), 12 (6 hrs), 9 (9 hrs), and 9 (24 hrs). h,i, Sesn^L431E animals have decreased food intake regardless of the leucine content of the food (h), and Sesn ^−/− animals have increased food intake regardless of the leucine content of the food (i). n=4 for all conditions. j, Whole-body re-expression of wild-type Sestrin driven by Tub>Gal4 is sufficient to partially restore the preference for leucine-containing food of Sesn ^−/− adult female flies. Animals with indicated genotypes were given the choice between leucine- or water- coated apples. Data show the preference of fold-difference. n (attP2) = 10, n (Sestrin WT) = 6. k, Adult female flies do not develop a preference for valine-containing apple regardless of their genotype. Animals with indicated genotypes were given the choice between leucine- or water- coated apples. Data show the preference of fold-difference. n (Wild type OreR, Sesn^L431E, Sesn^−/−) = 10, n (Wild type w¹¹¹⁸) = 12. Values are mean ± SD of technical replicates from a representative experiment. Data are representative of three independent experiments with similar results. Statistical analysis was performed using two-tailed unpaired t test (c, f, j), one-way ANOVA followed by Dunnett’s multiple comparisons test (d, g), one-way ANOVA followed by Tukey’s multiple comparisons test (e), two-way ANOVA followed by Tukey’s multiple comparisons test (h, i), and one-way ANOVA followed by Šídák’s multiple comparisons test (k).

Extended Data Fig. 5:. Leucine-sensing via the Sestrin-mTORC1 axis contributes to the detection of the protein content of food.

a, Wild-type (OreR) flies prefer food containing a high amount of yeast extract and this preference is reduced by the addition of leucine to food containing a low amount of yeast extract. Sesn^L431E flies have a reduced preference for the food containing a high amount of the yeast extract and the addition of leucine has minimal impact on the preference. How the food preference index was calculated is described in the methods. n (Wild type OreR, no leucine)=5, n (Wild type OreR, with leucine)=7, n (Sesn^L431E, no leucine)=6, n (Sesn^L431E, with leucine)= 9. b, As in (a) a choice experiment for wild type w¹¹¹⁸ and Sesn ^−/− flies. n (Wild type w¹¹¹⁸, no leucine)=9, n (Wild type w¹¹¹⁸, no leucine)=8, n (Sesn^−/−, no leucine)=9, n (Sesn^−/−, with leucine)= 12. Values are mean ± SD of technical replicates from a representative experiment. Data are representative of three independent experiments with similar results. Statistical analysis was performed using two-tailed unpaired t test, Holm-Šídák method.

Extended Data Fig. 6:. Flies prefer to lay eggs on leucine-containing food in a fashion that requires the leucine-binding capacity of Sestrin.

a, Schematic of the setup used in the egg-laying preference assay. Two identical apple pieces were painted with solutions containing different substances and placed on opposite sides of a container. Animals were allowed to feed ad libitum over the course of the assay and the number of eggs deposited on each apple was counted after 24 hours. b, c, Wild-type flies prefer to lay eggs on yeast- or amino acid-painted apples over water-painted apples. Scale bars, 1 mm. d-h, Sesn^L431E and Sesn ^−/− animals do not prefer to lay eggs on the leucine-containing apple. (a) created with BioRender.com. Values are mean ± SD of three technical replicates from a representative experiment. Data are representative of two independent experiments with similar results. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (d-g), and Šídák’s multiple comparisons test (h).

Extended Data Fig. 7:. Sestrin-regulated mTORC1 signaling in glial cells controls the preference of flies for leucine-containing food.

a, Same data as in Figure 4a except that the values were not normalized to the values from the flies expressing the control shRNA from each of the indicated drivers. n=5 (da, pros attP40 shRNA; da Sesn shRNA), 8 (repo, tdc2 attP40 shRNA; vGAT Sesn shRNA), 12 (repo, esg Sesn shRNA), 15 (Elav attP40 shRNA), 16 (Elav, Mef2, ddc Sesn shRNA), 22 (Mef2 attP40 shRNA; Myo1A Sesn shRNA), 10 (ddc, Lpp attP40 shRNA; tdc2, promE Sesn shRNA), 11 (vGAT attP40 shRNA; Lpp Sesn shRNA), 9 (promE attP40 shRNA; pros Sesn shRNA), 13 (esg attP40 shRNA), 24 (Myo1A attP40 shRNA). Each point represents the ratio of the amount of two oligonucleotide barcodes per 5 flies. b, Expression of wild-type Sestrin under repo-Gal4 driver in Sesn ^−/− flies is sufficient to partially rescue the leucine preference phenotype. n (repo-attP40 in wild type w¹¹¹⁸) = 4, n (other conditions) = 8. c, Overexpression of TSC1+TSC2 in glial cells using repo-Gal4; Tub-Gal80ts reduces the preference of flies for leucine-containing food. n (attP40) = 16, n (TSC1+2) = 19. d, The Sesn mRNA (red) is expressed in all classified subtypes of glial cells as indicated by co-expression of a pan glial marker, Repo (green). The single cell RNA sequencing dataset is from a previous study. e, The knock-down of Sestrin using a pan glial cell driver (repo-Gal4) reduces the leucine preference of flies much more significantly than a knockdown using drivers for glial subtypes. The knockdown of Sestrin in cortex glial cells using the wrapper-Gal4 driver line significantly decreased the leucine preference of flies. n=8 (repo, 9.GMR50A12, 15.R85G01-Gal4 attP40 shRNA; 9.GMR50A12, 15.R85G01-Gal4 Sesn shRNA), 12 (1.GMR60F04, 2.GMR53B07, 3.GMR55B03, 4.GMR56F03, 5.GMR86E01, 6.GMR53H12, 10.Alrm-Gal4 attP40 shRNA; repo, 2.GMR53B07, 3.GMR55B03, 4.GMR56F03, 5.GMR86E01, 10.Alrm-Gal4, 14.R75H03-Gal4 Sesn shRNA), 10 (7.GMR35E04 attP40 shRNA, 1.GMR60F04 Sesn shRNA), 11 (8.GMR77A03, 11.Wrapper-Gal4, 14.R75H03-Gal4 attP40 shRNA; 6.GMR53H12, 11.Wrapper-Gal4 Sesn shRNA), 28 (12.Eaat1-Gal4 39915, 13.Mdr65-Gal4 attP40 shRNA), 9 (7.GMR35E04, 8.GMR77A03 Sesn shRNA), 24 (12.Eaat1-Gal4 39915 Sesn shRNA), 18 (13.Mdr65-Gal4 Sesn shRNA). f, Confocal projection of wild-type female brains expressing 4MBOX-GFP fed the standard yeast-based food or starved of protein for 24 hours. Scale bar,10 μm. Values are mean ± SD of technical replicates from a representative experiment. Data are representative of two independent experiments with similar results. Statistical analysis was performed using two-tailed unpaired t test (a, c, e), and two-way ANOVA followed by Dunnett’s multiple comparisons test (b).

Extended Data Fig. 8:. Dietary leucine regulates mTORC1 signaling in glial cells in the peri-esophageal area in a fashion that depends on Sestrin and its capacity to bind leucine.

a, Schematic of the areas imaged and quantified for the ratio of GFP-positive cells to Repo-positive cells. The red rectangle represents zone 1, the orange rectangle represents zone 2, and the purple rectangle represents zone 3. b, Representative confocal images of zone 1 and zone 2 brain areas from wild-type, Sesn ^−/−, and Sesn^L431E female flies fed with an amino acid-replete or leucine-free diet. Scale bar, 25 μm. Note: images are reprocessed during revision from the same batch of samples as Figure 4c for the purpose of showing all zones 1, 2, and 3 clearly. The exact fly brains in the representative images and stacks might vary from Figure 4c, despite they are all from the same batch of samples. c, Representative confocal images of zone 3 brain areas of wild-type, Sesn ^−/−, and Sesn^L431E female flies fed an amino acid-replete or leucine-free diet. Scale bar, 10 μm. Note: images are from the same brains shown in (b). (a) created with BioRender.com. d, e, Quantification of the GFP-positive to Repo-positive ratio in zone 1 (d) and zone 3 (e). n=3 individual brains with indicated dietary treatment and genotype for each condition. Values are mean ± SD of biological replicates from a representative experiment. Data are representative of three independent experiments with similar results. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test.

Fig. 1:. Drosophila Sestrin binds GATOR2 and regulates mTORC1 in vivo in response to dietary leucine.

a, Data from an equilibrium binding assay showing that purified FLAG-Sestrin bound leucine, Kd~100 μM. Values are mean ± SD of three technical replicates from a representative experiment. b, The L431E mutation blocks leucine binding by Drosophila Sestrin. HA-tagged wild-type Sestrin and Sestrin(L431E) were prepared from HEK293T cells expressing the appropriate cDNAs. The binding assays were performed as in (a). Values are mean ± SD of three technical replicates from a representative experiment. The P values were determined using an unpaired t test with Welch correction, and the Holm-Šídák multiple comparison method. c, Leucine starvation enhances the Sestrin-GATOR2 interaction. FLAG-immunoprecipitates (IPs) were prepared from S2R+ cells stably expressing FLAG-tagged und (negative control) or WDR59 (a GATOR2 component) and starved or not of leucine. IPs and lysates were analyzed by immunoblotting for indicated proteins. Addition to the IPs of 1 mM of leucine, but not other amino acids, disrupted the Sestrin-GATOR2 interaction. d, Dietary leucine regulates in vivo the interaction of Sestrin with GATOR2 depending on the leucine-binding site of Sestrin. Immunoprecipitates were prepared from lysates of fat bodies from Wild-type (OreR) or Sesn^L431E larvae expressing the MYC-tagged control protein GFP or the MYC-tagged GATOR2 component WDR24 in the fat body (lpp-gal4). Animals were fed the indicated diets for 4.5 hours before sample collection. Amino-acid-replete: chemically defined diet containing all amino acids; leucine-free or valine-free: chemically defined diet lacking leucine or valine, respectively. e, Sestrin binding to leucine regulates mTORC1 signaling in vivo. Shown are immunoblots of Sestrin, S6K and phospho-S6K in fat bodies prepared as in (d) from larvae with indicated genotypes. Nprl2 and Mio encode core components of the GATOR1 and GATOR2 complexes, respectively. Dietary composition and feeding period were as in (d). Data are representative of three (a, b) or two (c-e) independent experiments with similar results.

Fig. 2:. Drosophila require Sestrin to adapt to a low-leucine diet.

a, b, Loss of Sestrin reduces survival during development upon leucine starvation. The bar charts show survival (%) of larvae raised for 10 days on a chemically defined diet containing 10% of the leucine in the control diet. The P values were determined using two-proportion z-test (two-sided). The bars show the percentage of surviving larvae in each genotype and the error bars represent the 95% Wald confidence interval. c, Sestrin is required for larval growth on a low-leucine diet. Shown are age-synchronized animals of the indicated genotypes raised 9 days on either an amino-acid-replete diet or a reduced (10%)-leucine diet. Scale bar, 1 mm. d-i, Loss of Sestrin reduces survival of adult flies upon leucine starvation. Sesn^−/− animals show reduced lifespan when fed a diet lacking leucine (0% leucine). Survival curves of age-synchronized adult male and female animals of the indicated genotypes fed the indicated diets. (c) nWT(w¹¹¹⁸)=157; nSesn^−/−=217; (d) nWT(w¹¹¹⁸)=221; nSesn^−/−=225; (e) nWT(w¹¹¹⁸)=206; nSesn^−/−=203; (f) nWT(w¹¹¹⁸)=205; nSesn^−/−=226; (g) nWT(w¹¹¹⁸)=222; nSesn^−/−=230; (h) nWT(w¹¹¹⁸)=221; nSesn^−/−=228. See statistics in Supplementary Data 1 and Methods. (a-i), Data are representative of three independent experiments with similar results.

Fig. 3:. Flies prefer to eat leucine-containing food in a fashion that depends on the capacity of Sestrin to bind leucine.

a, A schematic of the two-choice food preference assay (see Methods for details). AA, amino acids. b, Wild-type female animals develop a preference for leucine over the course of several hours. Data show the fold-difference in relative food intake for the leucine-coated compared to water-coated apples. n≥11 per time point. c, Rapamycin prevents flies from developing a preference for the leucine-coated apple. n≥5 per condition. d-f, Sesn^L431E and Sesn^−/− animals fail to develop a preference for the leucine-containing apple. (d, e), n≥4 per condition; (f) n≥6 per condition. g, Immunoblotting for Sestrin following knockdown of Sesn in adult flies. Akt serves as a loading control. h, Ubiquitous knockdown of Sesn reduces the preference of adult female flies for leucine. Data show the fold-difference in food intake for the leucine-coated apple relative to the water-coated apple. n≥5 per condition. i, The approach used to achieve temporal control of Sesn knockdown in (j, k). j, Sesn immunoblot showing Gal80^ts-mediated depletion of Sestrin in adult, but not developing, animals. Extracts were prepared from flies raised at indicated temperatures. S6K serves as a loading control. Note that heat shock induces Sestrin protein levels in control flies. k, Knockdown of Sestrin during adulthood is sufficient to decrease the preference of female flies for leucine-containing apples. n≥13 per condition. (a, i) created with BioRender.com. (b, c, d-f, h, k), Values are mean ± SD of biological replicates from a representative experiment. Each experiment was repeated three (d-k) or two (b, c) times with similar results. Statistical analyses: one-way ANOVA followed by Dunnett’s multiple comparisons test (b), two-way ANOVA followed by Šídák’s multiple comparisons test (c-e), one-way ANOVA followed by Šídák’s multiple comparisons test (f), and two-tailed unpaired t-test (h, k).

Fig. 4:. Sestrin-regulated mTORC1 signaling in glial cells controls the preference of flies for leucine-containing food.

a, A genetic screen identifies a role for Sesn in glial cells in mediating the leucine preference. Sesn RNA-mediated interference was performed in various tissues with the indicated Gal4 lines. Knockdown of Sesn in glial cells (Repo-Gal4) and ubiquitously (da-Gal4), but not in other tissues, reduces the preference for the leucine-coated versus water-coated apple. For each Gal4 line, data are normalized to the leucine preference of control flies. See non-normalized data in Extended Data Figure 7a. n≥5 per condition. b, c, Confocal projection of brains of adult female flies of the indicated genotypes expressing 4MBOX-GFP, a reporter for the MITF transcription factor that is negatively regulated by mTORC1. Animals were fed the indicated diets for 1 day and brains were stained for GFP and Repo. Images in (b) and (c) were taken with 10X and 40x objectives, respectively. Scale bars in (b) and (c) represent 50 μm and 10 μm, respectively. d, In wild-type flies, but not Sesn^L431E or Sesn^−/−flies, leucine starvation increases the number of GFP-positive peri-oesophageal glial cells. Each point represents the ratio of the number of GFP- to Repo-positive cells in the oesophageal area of one fly brain. n≥3 per condition. e, Proposed role of the Sestrin-mTORC1 pathway in regulating the preference of flies for leucine-containing food. (a, d), Values are mean ± SD of technical replicates from a representative experiment. Data are representative of three independent experiments with similar results. Statistical analysis was performed using two-tailed unpaired t test (a), and two-way ANOVA followed by Šídák’s multiple comparisons test (d).