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

Nature volume 608, pages 209–216 (2022)Cite this article

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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 deprivation2,3, 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 (SesnL431E) 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.

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Fig. 1: Drosophila Sestrin binds GATOR2 and regulates mTORC1 in vivo in response to dietary leucine.
Fig. 2: Drosophila require Sestrin to adapt to a low-leucine diet.
Fig. 3: Flies prefer to eat leucine-containing food in a fashion that depends on the capacity of Sestrin to bind leucine.
Fig. 4: Sestrin-regulated mTORC1 signalling in glial cells controls the preference of flies for leucine-containing food.

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Data availability

The data that support the findings of this study are available from the corresponding authors and the Whitehead Institute (sabadmin@wi.mit.edu) upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank all members of the laboratories of D.M.S. and N.P. for helpful insights and suggestions, as well as J. H. Lee, J. D. Levine, M. A. Lilly, F. Pignoni, A. A. Teleman, the Bloomington Drosophila Stock Center, and the Developmental Studies Hybridoma Bank for providing fly stocks and reagents. We thank P. Rosen for critical reading of the manuscript and R. Chivukula and T. Wang for experimental suggestions. We thank C. Lewis, B. Chan and T. Kunchok for performing the LC–MS analyses and L. Parel for help on mutant genotyping. This work was supported by grants from the NIH to D.M.S. (R01 CA103866 and R37 AI47389), N.P. (5P01 CA120964-04 and R01 AR057352), J.W.L. (R01 CA193256), M.L.V. (F30 CA228229 and T32 GM007753), A.E.A. (F31 CA232658), and N.K. (T32 HG002295), the Department of Defense (W81XWH-07-0448) to D.M.S., the American Cancer Society postdoctoral fellowship to M.A.R., the American Cancer Society Research Scholar Award to J.W.L., the Cystinosis Research Foundation to P.J. and N.P., and the Ludwig Center Graduate Fellowship to X.G. from the Koch Institute for Integrative Cancer Research at MIT. N.P. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Xin Gu, Patrick Jouandin

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Xin Gu, Pranav V. Lalgudi & Max L. Valenstein

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Xin Gu, Pranav V. Lalgudi & Max L. Valenstein

  3. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Patrick Jouandin, Rich Binari, Nolan Kamitaki & Norbert Perrimon

  4. Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Patrick Jouandin, Rich Binari & Norbert Perrimon

  5. Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA

    Michael A. Reid, Annamarie E. Allen & Jason W. Locasale

  6. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA

    Nolan Kamitaki

  7. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

    Nolan Kamitaki

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  1. Unaffiliated: David M. Sabatini

    • David M. Sabatini
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Contributions

X.G. and D.M.S. developed the research plan and together with P.J. and N.P. interpreted experimental results. X.G. and P.J. designed and performed all experiments. R.B. helped with ordering and maintaining fly stocks. M.L.V. and P.V.L. helped with experimental design and data analysis. N.K. helped with statistical analysis. M.A.R., A.E.A. and J.W.L. provided the recipe for the chemically defined food and prepared the first batches of it. X.G. and D.M.S. wrote the manuscript and P.J. and N.P. helped edit it.

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Correspondence to Xin Gu, Patrick Jouandin or Norbert Perrimon.

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Competing interests

D.M.S. is a shareholder of Navitor Pharmaceuticals, which is targeting for therapeutic benefit the amino-acid-sensing pathway upstream of mTORC1. J.W.L. advises Raphael Pharmaceuticals, Nanocare Technologies, Petri Biologics, and Restoration Foodworks. M.A.R. is currently employed by Amgen, which has interests in neurodegenerative diseases. These relationships have no overlap with this study. The other authors declare no competing interests.

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Extended data figures and tables

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 SesnL431E larvae fed the indicated diet for 4.5 h. Values are mean ± SD of biological 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 h. The Sesn RNAi was expressed in clones of cells (GFP, outlined) with a FLP-out system70. 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.

Source Data

Extended Data Fig. 2 The SesnL431E 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 curves for animals of the indicated sex and genotypes fed the indicated chemically-defined diets. (a) nWT(OreR)=235; n(SesnL431E)=238; (b) nWT(OreR)=233; n(SesnL431E)=237; (c) nWT(OreR)=242; n(SesnL431E)=248; (d) nWT(OreR)=242; n(SesnL431E)=240; (e) nWT(OreR)=229; n(SesnL431E)=243; (f) nWT(OreR)=245; n(SesnL431E)=245. See statistics in Supplementary Data 1 and methods. h. SesnL431E 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).

Source Data

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 h 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 h. Results are quantified in (e). Scale bar, 500 μm. f, g, SesnL431E flies have reduced fecundity but not fertility. (f) Number of eggs laid over a period of 60 h 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 biological replicates from a representative experiment. (e) n = 6 (Wild type (w1118)), 8 (Sesn−/−), 7 (SesnL431E amino acid-replete diet), 5 (SesnL431E leucine-free diet), and 6 (SesnL431E 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.

Source Data

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 described27. 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, SesnL431E animals do not have a preference for valine- over water-painted apples. 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, SesnL431E 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, SesnL431E, Sesn−/−) = 10, n (Wild type w1118) = 12. 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-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).

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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. SesnL431E 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 (SesnL431E, no leucine)=6, n (SesnL431E, 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 biological 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.

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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 h. 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, SesnL431E 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 biological 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).

Source Data

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 study39. 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 h. Scale bar, 10 μm. Values are mean ± SD of biological 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).

Source Data

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 SesnL431E 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 SesnL431E 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.

Source Data

Extended Data Table 1 Chemically defined food “Part 1” mixture
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Extended Data Table 2 Chemically defined food “Part 2” mixture
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Extended Data Table 3 Amino acid stock solutions
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Extended Data Table 4 Other stock solutions
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Supplementary information

Supplementary Information

Raw western blots with figure panels labelled.

Reporting Summary

Supplementary Data 1

This file contains data for Fig. 2 and Extended Data Fig. 5.

Supplementary Data 2

Pupariation data analysis code.

Supplementary Data 3

Survival data analysis R script.

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Gu, X., Jouandin, P., Lalgudi, P.V. et al. Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila. Nature 608, 209–216 (2022). https://doi.org/10.1038/s41586-022-04960-2

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