Abstract
A balanced intake of macronutrients—protein, carbohydrate and fat—is essential for the well-being of organisms. An adequate calorific intake but with insufficient protein consumption can lead to several ailments, including kwashiorkor1. Taste receptors (T1R1–T1R3)2 can detect amino acids in the environment, and cellular sensors (Gcn2 and Tor)3 monitor the levels of amino acids in the cell. When deprived of dietary protein, animals select a food source that contains a greater proportion of protein or essential amino acids (EAAs)4. This suggests that food selection is geared towards achieving the target amount of a particular macronutrient with assistance of the EAA-specific hunger-driven response, which is poorly understood. Here we show in Drosophila that a microbiome–gut–brain axis detects a deficit of EAAs and stimulates a compensatory appetite for EAAs. We found that the neuropeptide CNMamide (CNMa)5 was highly induced in enterocytes of the anterior midgut during protein deprivation. Silencing of the CNMa–CNMa receptor axis blocked the EAA-specific hunger-driven response in deprived flies. Furthermore, gnotobiotic flies bearing an EAA-producing symbiotic microbiome exhibited a reduced appetite for EAAs. By contrast, gnotobiotic flies with a mutant microbiome that did not produce leucine or other EAAs showed higher expression of CNMa and a greater compensatory appetite for EAAs. We propose that gut enterocytes sense the levels of diet- and microbiome-derived EAAs and communicate the EAA-deprived condition to the brain through CNMa.
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Data availability
The original data for the immunoblotting are shown in Supplementary Fig. 1. All other raw data are available from corresponding authors on request. Source data are provided with this paper.
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Acknowledgements
We thank the W.-J.L. and G.S.B.S. laboratory members for discussion and critical reading of the manuscript, and we thank the KAIST Biocore center (operator: J. Kim). This work is supported by grants from the Samsung Science and Technology Foundation (project number: SSTF-BA-1802-11) and the National Research Foundation of Korea (NRF-2020R1A2C2009865); by NIH R01 grants (R01DK116294 and R01DK106636, which have been transferred to H. Don Ryoo at New York University) to G.S.B.S; and by grants from National Creative Research Initiative programs of the National Research Foundation of South Korea (2015R1A3A2033475) and the Samsung Science and Technology Foundation (project number: SSTF-BA-1401-15) to W.-J.L. B.K. is supported by the National Research Foundation of South Korea (NRF-2020R1I1A1A01072255).
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B.K. and M.I.K. conceived, designed, performed and analysed the experiments. Y.O., M.K., E.-K.K., I.-H.J., J.-H.L. and S.-G.K. performed the experiments. G.S.B.S. and W.-J.L. conceived and supervised the project and designed and analysed the experiments. G.S.B.S., W.-J.L. and B.K. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Compensatory appetite for EAAs in amino-acid-deprived male flies, virgin female flies, poxn-mutant flies and mated female flies in which Ir76b+ neurons or Dh44+ neurons were inactivated.
a–c, Two-choice preferences of wild-type w1118 mated female (a), male (b) and virgin female (c) flies that had been fed (−) or had been deprived of amino acids for the indicated durations (5, 24, 48 and 72 h). Flies were given a choice between: l-EAA + l-NEAA + d-glucose and d-EAA + d-NEAA + d-glucose (a) or l-EAA + d-glucose and d-EAA + d-glucose (b, c). d, Food preferences of w1118 mated female flies fed with diets containing different amounts of dietary yeast were given a choice between l-EAA + d-glucose and d-EAA + d-glucose. e, Food preferences of flies in which Ir76b+ neurons had been inactivated by expressing tetanus toxin (TNT) (Ir76b-LexA>LexAop-TNT flies) were fed (−) or were deprived of amino acids for 48 h before the two-choice assay: l-EAA + d-glucose versus d-EAA + d-glucose. Flies carrying Ir76b-LexA alone or LexAop-TNT alone were used as controls. f, Two-choice preferences of control w1118 and poxn−/− flies that had been deprived of amino acids for 48 h. g, Two-choice preferences of flies carrying Dh44-Gal4 and UAS-Kir2.1 that had been fed (−) or had been deprived of amino acids for 48 h. Flies carrying Dh44-Gal4 alone or UAS-Kir2.1 alone were used as controls. Inactivation of Dh44+ neurons was achieved by expressing inwardly rectifying potassium channels (Kir2.1) in Dh44+ neurons. Data are mean ± s.e.m. P values are indicated; unpaired two-tailed t-test in a; one-way ANOVA with Tukey’s post-hoc test in b–g. n is experimental trials. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 2 Amino acid deprivation enhances the expression of CNMa in enterocytes, but not in intestinal stem cells, enteroblasts or enteroendocrine cells.
a, Normalized CNMa mRNA expression levels measured by qPCR in flies that were fed isocaloric diets containing varying amounts of dietary yeast for 5–6 days (n = 5). b, c, CNMa promoter activity in the gut of CNMa-GAL4>UAS-GFP flies that were fed an isocaloric diet containing 2% (low-amino-acid diet) versus 10% (high-amino-acid diet) of yeast for 7 days. Representative confocal images of the whole gut (b) and quantifications of the GFP fluorescence in the R2 segment of the anterior midgut (c) are shown. d, Representative confocal images showing enterocytes in the anterior R2 midgut that express CNMa. Intestinal stem cells (ISCs) and enteroblasts (EBs) were respectively marked by β-galactose (β-gal) staining of the gut in flies carrying Delta-lacZ (red), CNMa-Gal4 and UAS-GFP (green) (top left), and in flies bearing Su(H)-lacZ (red), CNMa-Gal4 and UAS-GFP (green) (top middle). Enteroendocrine cells (EEs) are labelled by anti-prospero antibody staining (red) (top right), whereas enterocytes (ECs) are identified on the basis of their large polyploidy nuclei as revealed by DAPI staining (blue). In all cases, CNMa expression was visualized by GFP (green). A schematic representation of different intestinal cell types is shown. e, f, Representative confocal images of enterocytes in the R2 (e) and R4 (f) midgut regions of a fly carrying NP1-Gal4 (enterocyte-specific driver) and UAS-CD8-GFP co-labelled with anti-GFP (green) and anti-CNMa (red) antibodies. Nuclei are stained with DAPI (blue). Scale bars, 200 μm (b); 50 μm (c); 10 μm (d–f). Single-plane images are shown. Representative images are selected from at least 20 independent samples collected in each experiment. Data are mean ± s.e.m. P values are indicated; one-way ANOVA with Tukey’s post-hoc test in a; unpaired two-tailed t-test in c. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 3 Amino acid deprivation has no effect on CNMa expression in the fat body or the brain, whereas EAA deprivation but not NEAA deprivation influences CNMa expression in the gut.
a, b, Representative confocal images (left), the relative signal intensity of native GFP fluorescence (middle) and the numbers of GFP-positive cells (right) in the fat body (a) and the brain (b; insets show the dorsal region) in flies carrying CNMa-Gal4 and UAS-GFP (green) that were fed a high-amino-acid versus a low-amino-acid diet. Nuclei in the fat body counterstained with DAPI (a, left), and the brain stained with anti-GFP antibody (green) and the neuropil marker nc82 (magenta) (b, left) are shown. The numbers of GFP-positive cells are as follows: 41.00 ± 7.28 (high amino acids) and 41.54 ± 8.16 (low amino acids) in the fat body; 3.96 ± 1.48 (high amino acids) and 3.29 ± 1.33 (low amino acids) in the brain. Scale bars, 50 μm (a, b); 10 μm (b, inset). c, d, EAA deprivation, but not NEAA deprivation, induced CNMa expression (c) and possibly mobilized intracellular calcium (d). Quantifications of GFP fluorescence in the R2 region of the anterior midgut in CNMa-GAL4>UAS-GFP flies (c) or CNMa-GAL4>CaLexA flies (d) that were fed with an EAA-deficient versus a NEAA-deficient diet for two days. e, f, Deficiency of a single EAA in a diet induced CNMa expression (e) and possibly mobilized intracellular calcium (f). Quantifications of GFP fluorescence in the R2 region in CNMa-GAL4>UAS-GFP flies (e) or CNMa-GAL4>CaLexA flies (f) that were fed with a holidic diet lacking a single EAA for three days. Data are mean ± s.e.m. P values are indicated; unpaired two-tailed t-test in a, b; one-way ANOVA with Tukey’s post-hoc test in c–f. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 4 Molecular characterization of CNMa and CNMaR knockout lines.
a, b, The genomic loci of CNMa (a) and CNMaR (b) before and after the deletions were generated. The open reading frames and the cleavage sites used by gRNAs are indicated by yellow boxes and arrowheads, respectively. The gRNA-matching sequences are denoted in bold. Deleted regions were confirmed by sequencing of the genomic DNA; 20 base pairs (AGGAAGGAAGGCGATGGATT) and 2 base pairs (AC) were inserted in the deletion region of CNMa and CNMaR loci, respectively. c, The PCR analyses confirmed the lack of CNMa and CNMaR transcripts in CNMa−/− and CNMaR−/− flies, respectively. Representative images were selected from three independent samples collected in each experiment.
Extended Data Fig. 5 The activities of Tor and Gcn2 signalling pathways modulated by EAA deprivation regulate CNMa expression in the gut, but have no effect on CNMa expression in the brain or fat body.
a, Two-choice behavioural preferences of amino-acid-deprived flies in which UAS-CNMaRNAi had been expressed specifically in the enterocytes (NP1-Gal4), fat body (Cg-Gal4) or neurons (Nsyb-Gal4). Flies carrying tissue-specific GAL4 alone were used as controls. b, c, Western blot analysis of phosphorylated eIF2α (p-eIF2α) and phosphorylated ribosomal protein S6K (p-S6K) in whole-gut lysates collected from mated female flies (around 5–6-days old) that were deprived of EAA (−EAA) for 48 h (b). The intensity of each band corresponding to phosphorylated eIF2α and S6K was quantified using the ImageJ automated digitizing program (NIH) (c). Results from three independent experiments were normalized against those in control flies, the average of which is arbitrarily set as 1. d, Quantifications of GFP fluorescence in the R2 region of the anterior midgut of CNMa-Gal4>UAS-GFP flies (control) or control flies carrying either UAS-mRFP, UAS-ninaBRNAi, UAS-Gcn2RNAi, UAS-Atf4RNAi, UAS-TorWT or UAS-MitfRNAi. e, f, Levels of CNMa expression in the brain or fat body of flies carrying CNMa-Gal4 and UAS-GFP that also harboured either UAS-TorWT or UAS-Gcn2RNAi. GFP signal intensities in the fat body (e) or brain (f) were measured by the native GFP fluorescence. Data are mean ± s.e.m. P values are indicated; unpaired two-tailed t-test in a, c; one-way ANOVA with Tukey’s post-hoc test in d–f. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 6 Enhancement of the CNMa promoter activity by co-expression of Atf4 and Mitf.
The 5′-flanking region (−1573/+1) of the CNMa gene was used to generate a pGL3-CNMa reporter plasmid. Drosophila S2 cells were transfected with the pGL3-CNMa reporter plasmid with pPac-Atf4 (Atf4), pPac-Mitf (Mitf) or both (Atf4 + Mitf). Luciferase activities were measured at 48 h after transfection. The luciferase activity of the pGL3-CNMa reporter co-transfected with an empty pPac vector was arbitrarily set to 1. The relative luciferase activities are shown as the mean ± s.e.m. from three independent experiments. P values are indicated (one-way ANOVA with Tukey’s post-hoc test). Statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 7 CNMaR-expressing neurons are located in the anterior midgut and the brain and are not overlapped with Ir76b+ neurons, and some respond to amino acid deprivation.
a, b, Representative confocal images of the anterior midgut of an adult fly carrying CNMaR-Gal4 and UAS-CD8-GFP that were co-labelled with anti-GFP antibody (green) and DAPI (blue) (a), and the brain of an adult fly carrying Ir76b-Gal4>UAS-CD8-GFP and CNMaR-LexA>LexAop-tdTomato, probed with anti-GFP (green) and anti-DsRed (red) antibodies, illustrate two populations of neuronal processes: one from Ir76b+ taste sensory neurons and another from CNMaR+ central nervous system (CNS) neurons (b). c, Measurements of calcium mobilization by CaLexA reporter (green) in mated female flies carrying CNMaR-Gal4>UAS-CaLexA (flies of around 2 days old maintained for 5 days on a high-amino-acid diet containing 10% dietary yeast) that were subjected to amino acid deprivation by transferring half of the flies to an agar medium containing 100mM d-glucose only for 48 h (right, protein deprivation) or that continued on the high-amino-acid diet (left, no deprivation). Arrowheads denote the enhancement of GFP intensity in the ellipsoid body (right). Scale bars, 200 μm (a); 50 μm (b, c). Z-stacked projection images are shown. Representative images were selected from at least five independent samples collected in each experiment.
Extended Data Fig. 8 Commensal bacteria influence CNMa expression in the gut, but not in the fat body or brain.
a, Quantifications of GFP fluorescence in the R2 region of the gut of conventionally reared and GF female flies carrying CNMa-Gal4>UAS-GFP fed on 10% or 15% dietary yeast. b, c, The level of CNMa expression in the fat body (b) or the brain (c) of flies bearing CNMa-Gal4 and UAS-GFP that had been reared in 10% dietary yeast were quantified using the GFP signal intensity measured by its native GFP fluorescence. d, Quantifications of GFP fluorescence in the R2 region of GF flies that had been mono-associated with A. pomorum or L. plantarum WJL. Data are mean ± s.e.m. P values are indicated; one-way ANOVA with Tukey’s post-hoc test in a, d; unpaired two-tailed t-test in b, c. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 9 Acetobacter colonization results in a significant increase in the amino acid absorption of the host and the suppression of the l-EAA preference through CNMaR-expressing neurons.
a, Levels of ingested deuterium-labelled amino acids (l-leucine-5,5,5-d3, l-phenyl-d5-alanine-2,3,3-d3 or l-glutamic acid-2,3,3,4,4-d5) in the haemolymph of GF control flies (−) fed on 5% sucrose alone and in the haemolymph of treated GF flies (+) fed on 5% sucrose containing deuterium-labelled amino acids (n = 3). b, Two-choice preferences of GF flies or GF flies that had been mono-associated with A. pomorum in which CNMaR+ neurons were conditionally activated by the heat-inducible TrpA1 at 30 °C. Note that a significantly higher bacterial load (approximately 10 times higher number of colony-forming units) of intestinal A. pomorum was found in flies maintained at 30 °C than in flies maintained at 22 °C in which control experiments were conducted (data not shown). GF-Aceto, GF flies mono-associated with A. pomorum; GF-Lacto, GF flies mono-associated with L. plantarum WJL. Data are mean ± s.e.m. P values are indicated; one-way ANOVA with Tukey’s post-hoc test in a, b. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Extended Data Fig. 10 CNMa expression and l-EAA preference are regulated by the microbiome and Tor signalling in the gut.
a, Quantifications of GFP fluorescence in the R2 region of the gut of GF flies carrying CNMa-Gal4>UAS-GFP that had been mono-associated with Acetobacter that has a mutation in the proC gene (AcetoΔproC) or the leuB gene (AcetoΔleuB). Complementation of the AcetoΔleuB strain was achieved by re-introducing the leuB gene to generate AcetoΔleuB_leuB. Dietary complementation of AcetoΔleuB was achieved by adding 10 mM leucine to the diet. b, c, Representative confocal images (left) and quantifications of GFP fluorescence (right) in the R2 region of the gut (b) and two-choice preferences (c) of GF flies that had been mono-associated with Acetobacter that has a mutation in ilvA (AcetoΔilvA). Dietary complementation of AcetoΔilvA was achieved by adding 10 mM isoleucine to the diet. GF flies carrying CNMa-Gal4>UAS-GFP and GF w1118 flies were used in b and c, respectively. d, Quantifications of GFP fluorescence in the R2 region of the gut of GF flies carrying CNMa-Gal4>UAS-GFP that had been mono-associated with an engineered strain of L. plantarum WJL that is capable of producing BCAAs (LactoBCAA). Dietary complementation of L.plantarum WJL (LactoWT) was achieved by adding 10 mM of leucine, isoleucine and valine to the diet (LactoWT + BCAA). e, Representative confocal images (left) and quantifications of the GFP signal (right) in the anterior R2 midgut region of AcetoΔleuB mono-associated GF flies carrying CNMa-Gal4 and UAS-GFP (GFcontrol), and flies carrying CNMa-Gal4, UAS-GFP and UAS-TorWT (GFTor-WT) on 10% dietary yeast, measured by the native GFP fluorescence. f, Two-choice preferences of AcetoΔleuB mono-associated flies carrying enterocyte-specific NP1-Gal4 (control), and flies carrying NP1-Gal4 and UAS-TorWT (n = 20). Nuclei were stained with DAPI. Scale bars, 50 μm (b, e). Data are mean ± s.e.m. P values are indicated; one-way ANOVA with Tukey’s post-hoc test in a–d; unpaired two-tailed t-test in e, f. Sample sizes and statistical analyses are shown in Supplementary Table 1.
Supplementary information
Supplementary Figure 1
Uncropped images of Western blot (related to Extended Data Figure 5b).
Supplementary Tables
This file contains Supplementary Tables 1-4. Supplementary Table 1 contains detailed descriptions of the experimental conditions, sample sizes, and statistical analyses that were used in this study. Supplementary Table 2 contains food sources for two choice assay that were used in this study. Supplementary Table 3 contains real-time qPCR primer sequences that were used in this study (related to Figure 1d and Extended Data Figure 2a). Supplementary Table 4 contains PCR primer sequences that were used in this study (related to Extended Data Figure 4c).
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Kim, B., Kanai, M.I., Oh, Y. et al. Response of the microbiome–gut–brain axis in Drosophila to amino acid deficit. Nature 593, 570–574 (2021). https://doi.org/10.1038/s41586-021-03522-2
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DOI: https://doi.org/10.1038/s41586-021-03522-2
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