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Sensing of the non-essential amino acid tyrosine governs the response to protein restriction in Drosophila

Abstract

The intake of dietary protein regulates growth, metabolism, fecundity and lifespan across various species, which makes amino acid (AA)-sensing vital for adaptation to the nutritional environment. The general control nonderepressible 2 (GCN2)-activating transcription factor 4 (ATF4) pathway and the mechanistic target of rapamycin complex 1 (mTORC1) pathway are involved in AA-sensing. However, it is not fully understood which AAs regulate these two pathways in living animals and how they coordinate responses to protein restriction. Here we show in Drosophila that the non-essential AA tyrosine (Tyr) is a nutritional cue in the fat body necessary and sufficient for promoting adaptive responses to a low-protein diet, which entails reduction of protein synthesis and mTORC1 activity and increased food intake. This adaptation is regulated by dietary Tyr through GCN2-independent induction of ATF4 target genes in the fat body. This study identifies the Tyr–ATF4 axis as a regulator of the physiological response to a low-protein diet and sheds light on the essential function of a non-essential nutrient.

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Fig. 1: Low-protein diet suppresses protein synthesis in ATF4-dependent manner.
Fig. 2: Tyr supplementation to a low protein diet suppressed ATF4 activity and protein synthesis.
Fig. 3: Tyr restriction induces ATF4-dependent 4E-BP induction.
Fig. 4: Dietary Tyr restriction decreases internal Tyr levels and induces 4E-BP.
Fig. 5: Tyr restriction induces 4E-BP independently of GCN2 and eIF2α.
Fig. 6: Tyr–ATF4–scyl axis regulates mTORC1 activity during protein restriction.
Fig. 7: Tyr scarcity increases CNMa in the fat body which is received in the serotonergic neurons.
Fig. 8: CNMa and its receptor regulate adaptive increase of food intake.

Data availability

The NGS data are available under accession nos. DRA013060, DRA013061, DRA009291 and DRA010710. The data used to analyze the results in this paper are available as source data. All the materials generated in this study are available upon request to F.O. Source data are provided with this paper.

Code availability

No custom codes were used during this study.

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Acknowledgements

We acknowledge A. P. Gould (the Francis Crick Institute), H. D. Ryoo (New York University School of Medicine), W.J. Lee (Seoul National University), R. Carthew (Northwestern University), O. V. Alekseyenko (Harvard Medical School), the Kyoto Stock Center, National Institute of Genetics, Vienna Drosophila Resource Center and Bloomington Drosophila Stock Center for reagents. We thank S. Sorge, A. Franchet, L. Lampe, A. P. Gould and Y. Yoshinari for critical comments on the manuscript. We thank T. Fujisawa, T. Ichinose, H. Tanimoto and all members of our laboratory for technical assistance and critical advice. We appreciate the generous support of Shimadzu, which provided the LC–MS platform for the Suematsu laboratory but did not participate in designing the study or analyzing the data. This work was supported by AMED-PRIME to F.O. under grant nos. JP17gm6010010 and JP20gm6310011 and by AMED-Project for Elucidating and Controlling Mechanisms of Aging and Longevity to M.M under grant no. JP21gm5010001. This work was also supported by grants from the Japan Society for the Promotion of Science to F.O. under grant nos. 19H03367, 20H05726 and 22H02769 and to M.M. under grant nos. 16H06385, 21H04774 and 21K19206. This work was partially supported by the Uehara Memorial Foundation to F.O., the Tomizawa Jun-ichi & Keiko Fund of the Molecular Biology Society of Japan for Young Scientists to N.O. and F.O. and the Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan. H.K. is a JSPS research fellow.

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Contributions

H.K. and F.O. conceived the project. H.K. performed most of the experiments and analyzed the data. N.O. and R.N. analyzed the CNMaR expression pattern. H.A. analyzed bacterial composition and helped with systematic analysis of the NEAA restrictions. Y.S. and M.S. performed some of the metabolome analyses. H.K., N.O., R.N., M.M. and F.O. wrote the initial manuscript. M.M. and F.O. supervised the study. All authors edited and approved the final manuscript.

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Correspondence to Fumiaki Obata.

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Nature Metabolism thanks Matthew Piper and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling editor: Yanina-Yasmin Pesch, Ashley Castellanos-Janciewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Internal AA levels during protein restriction.

a-d, Internal Tyr and Phe levels in the fat body (a, b) or hemolymph (c, d) of third instar larvae fed with a low-protein diet supplemented with or without 5 mM Tyr for 8 h. n = 4. e,f, Internal AA levels in the fat body (e) or hemolymph (f) of third instar larvae fed with a low-protein diet for 8 h. n = 4. For all graphs, Mean and SEM with all data points of biological replicates were shown. P-values are determined by one-way ANOVA with Holm-Šídák’s multiple comparison test (a-d) and unpaired two-tailed Student’s t-test (e,f). Asterisks indicate *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, ns, not significant.

Source data

Extended Data Fig. 2 Tissue specific transcriptional responses upon Tyr restriction.

a, Number of differentially expressed genes (Benjamini–Hochberg adjusted p-value < 0.01 after Wald test) in the gut or the fat body of third instar larvae during Tyr restriction. The numbers of either up- (log2 fold change > 0.59) or down- (log2 fold change < −0.59) regulated genes are shown. b, Quantitative RT-PCR of 4E-BP in the gut of third instar larvae during Tyr restriction. n = 9. P-value is determined by unpaired two-tailed Student’s t-test. c,d, Representative images of the fat body (c) or the other organs (d) of the ATF4 reporter 4E-BPintron-dsRed with or without Tyr restriction for 28 hours. Scale bars, 1 mm for the gut, 100 μm for the fat body, the salivary gland and the carcass. Ctrl indicates a complete holidic medium. For the graph, Mean and SEM were shown. Data points indicate biological replicates. ns, not significant. The experiments were repeated independently at least twice with similar results (c, d).

Source data

Extended Data Fig. 3 Phenotypic and metabolic analysis upon amino acid restrictions.

a, Fecundity of female flies upon AA restriction for four days. n = 8. Any EAA restrictions significantly (p < 0.0001) suppressed egg laying, while any NEAA did not. b, Body weight of third instar larvae upon Tyr or Leu restriction for 24 hours. n = 28 (Tyr-), 37 (Leu1/2), 30 (Leu1/4), 31 (Leu-). c, Whole body Tyr levels in female flies upon Tyr restriction for four days. n = 6. d,e, Whole body NEAA (d) or EAA (e) levels in adult male flies upon each NEAA (d) or EAA (e) restriction for four days. n = 6. f, Whole body Tyr metabolite levels in the third instar larvae upon Tyr restriction. n = 4. g, Whole body amount of Arg levels in the third instar larvae upon Arg restriction for 8 h. n = 6. Ctrl indicates a complete holidic medium. For all graphs, Mean and SEM were shown. Data points indicate biological replicates. P-values are determined by one-way ANOVA with Dunnett’s multiple comparison test (a), one-way ANOVA with Holm-Šídák’s multiple comparison test (b) and unpaired two-tailed Student’s t-test (c-g). Asterisks indicate *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, ns, not significant.

Source data

Extended Data Fig. 4 Contribution of gut microbiota to Tyr restriction-induced responses.

a, 16S rRNA amplicon sequencing analysis of the third instar larval gut microbiome during Tyr restriction. b,c, Quantitative PCR analysis of Lactobacillus (b) and Acetobacter (c) in the third instar larval gut during Tyr restriction. n = 4. d, Whole body NEAA levels in the germ-free larvae upon Ala, Tyr, or Asn restriction. n = 4 except for Tyr levels upon control diet (n = 3). e, Quantitative RT-PCR of 4E-BP in the larval fat body upon single NEAA restrictions for 8 h in germ-free condition. n = 6. Ctrl indicates a complete holidic medium. For all graphs, Mean and SEM with all data points of biological replicates were shown. P-values are determined by unpaired two-tailed Student’s t-test (b-d) and one-way ANOVA with Holm-Šídák’s multiple comparison test (e). Asterisks indicate *p < 0.05, ***p < 0.001 and ****p < 0.0001, ns, not significant.

Source data

Extended Data Fig. 5 Suppression of mTORC1 activity upon Tyr or Leu restriction.

a, b, A representative image (a) and quantification (b) of western blot analysis of the fat body using anti-phospho-S6 antibody upon Tyr or Leu restriction (1/2, 1/4 and complete depletion) for 8 h. Ctrl indicates a complete holidic medium. Anti-α+β tubulin was used for loading control. n = 3. P-value is determined by one-way ANOVA with Dunnett’s multiple comparison test. For the graph, Mean and SEM with all data points of biological replicates were shown. Asterisks indicate ****p < 0.0001.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 5–7

Reporting Summary

Supplementary Table 1

DEG list of fat body RNA-seq upon protein restriction.

Supplementary Table 2

Gene Ontology analysis of fat body RNA-seq upon protein restriction.

Supplementary Table 3

DEG list of fat body RNA-seq upon Tyr restriction.

Supplementary Table 4

DEG list of gut RNA-seq upon Tyr restriction.

Source data

Source Data Fig. 1

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Unprocessed western blots for Extended Data Fig. 5.

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Kosakamoto, H., Okamoto, N., Aikawa, H. et al. Sensing of the non-essential amino acid tyrosine governs the response to protein restriction in Drosophila. Nat Metab 4, 944–959 (2022). https://doi.org/10.1038/s42255-022-00608-7

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