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A nutrient-sensitive enterokine coordinates developmental plasticity through inter-organ signaling [1]

['Longwei Bai', 'Centre National De La Recherche Scientifique', 'Ecole Normale Supérieure De Lyon', 'Institut De Génomique Fonctionnelle De Lyon', 'Lyon', 'Jacques Montagne', 'Cathy Isaura Ramos', 'Université Claude Bernard Lyon', 'François Leulier']

Date: 2026-08 

Animal survival in fluctuating environments depends on the ability to modulate their developmental pace in response to nutrient availability, a phenomenon known as developmental plasticity. In Drosophila larvae, we uncover a critical endocrine mechanism that coordinates this process under conditions of amino acid restriction. We identify the peptide hormone Limostatin as an enterokine, produced by a small population of larval midgut enteroendocrine cells, that acts systemically to inhibit the expression and release of dIlp2, a major insulin-like peptide controlling developmental progression. Limostatin expression and secretion by enteroendocrine cells is triggered by reduced amino acid availability through an inter-organ relay involving the fat body and neuroendocrine insulin-producing cells in the brain. In turn, Limostatin participates in a feedback control loop that slows down developmental progression once systemic nutrient shortage is sensed. This bidirectional gut–brain axis enables larvae to preserve viability under nutritional stress. Our findings define the larval gut as a nutrient-sensitive endocrine organ and position Limostatin as a key regulator of developmental plasticity. Our work expands the concept of decretins to include developmental pace control, suggesting that enterokines that regulate IGF signaling, rather than insulin release per se, may represent an evolutionarily conserved or convergent strategy in regulating developmental plasticity.

Funding: This work was funded by Agence National de la Recherche ( https://anr.fr/en/ ) ANR-23-CE14-0034 (to F.L), Fondation de la Recherche Médicale ( https://www.frm.org/en ) FRM DEQ20180839196 (to F.L), Université Claude Bernard Lyon1 ( https://www.univ-lyon1.fr/en ) Bonus Qualité Recherche (to C.I.R) and Fondation Innovation en Infectiologie ( https://www.finovi.org/en:start ) FINOVI AO12-19-Projet SNI (to C.I.R). LB was funded by a scholarship from the Chinese Scholarship Council (CSC) of the Chinese government. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2026 Bai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Previous work identified the peptide hormone Limostatin (Lst) as a fasting-induced “decretin” hormone that suppresses dIlp production and release in adult flies [ 27 ]. Lst is produced by the corpora cardiaca, an endocrine tissue producing adipokinetic hormone (AKH)—the insect analog of glucagon—and its upregulation under sugar deprivation contributes to reduced systemic insulin signaling and increased glycemia. Although overexpression of Lst in the larval fat body was reported to delay development, Lst loss-of-function animals do not display overt developmental timing phenotype under standard laboratory conditions [ 27 ], raising questions about its developmental role and tissue-specific function. Here, we identify a novel, developmentally relevant role for Lst as a gut-derived hormone that controls developmental pace and survival in response to nutritional challenges. We show that Lst is specifically induced in a discrete group of enteroendocrine cells (EECs) in the anterior midgut of larvae exposed to extreme amino acid stress or milder amino acid stress coupled with microbiota depletion. We show that Lst production and release by EECs is induced upon reduced circulating dIlp2 levels, and that Lst in turn suppresses dIlp2 release from brain IPCs. This establishes an amplifying regulatory loop that reinforces the adaptation of developmental pace to amino acid scarcity. These findings establish that in contrast to its role as a decretin regulating sugar homeostasis in adults, Lst in juveniles acts as an enterokine, a gut-derived peptide hormone that integrates amino acid signals of dietary and microbial origins to coordinate developmental plasticity.

Drosophila melanogaster offers a genetically tractable model to dissect how these signals coordinate developmental pace with nutrient availability. Larval development in Drosophila is highly plastic, tightly regulated by environmental conditions, and largely orchestrated by insulin-like peptides (dIlps), which function as the principal somatotropic signals in insects [ 10 , 11 ]. dIlps can be produced locally [ 12 ] by a cluster of Insulin-producing cells (IPCs) in the brain, which act systemically through the insulin receptor (InR) in peripheral tissues [ 13 ]. One key downstream target of InR signaling is the transcription factor FoxO, which represses anabolic gene programs when dIlp signaling is reduced [ 14 , 15 ]. The output of IPCs’ activity is tightly regulated by a network of inter-organ communications [ 16 ]. Nutrient uptake by enterocytes in the gut, amino acid sensing via TORC1 in the fat body—a tissue that fulfils hepatic and adipose functions [ 17 ]—and fat body-derived adipokine signals [ 18 ] including Upd2 [ 19 ], Stunted [ 20 ], Eiger [ 21 ] and GBP1/2 [ 22 ] fine-tune dIlp production by IPCs to match nutritional status. Alongside these systemic cues, the intestinal microbiome contributes to developmental pace by producing limiting nutrients [ 23 ], facilitating digestion [ 24 ], and influencing endocrine pathways, including dIlps signaling [ 25 , 26 ]. Together, these findings highlight the complex cross-talk between diet, microbiota, and endocrine organs in shaping developmental growth and maturation.

Animals must continuously adjust their physiology to survive in environments where nutrient availability fluctuates, and a central aspect of this adaptation is the modulation of developmental pace in response to nutritional input, a process known as developmental plasticity [ 1 ]. Among dietary components, protein availability is a major determinant of somatic growth and maturation rate [ 2 ], and protein restriction typically triggers a reduction in growth rate and delayed maturation, an energy-conserving response that helps preserving developmental homeostasis to ensure survival to the adulthood minimizing costs on reproductive capacity [ 3 , 4 ]. In parallel, the intestinal microbiome plays a key role in digestion, nutrient assimilation and endocrine maturation, and is itself sensitive to dietary deprivation [ 5 , 6 ]. Alterations in microbial composition can either buffer or amplify the impact of poor nutrition by modulating host metabolism and hormonal signaling [ 7 , 8 ]. Thus, developmental plasticity is best understood as a multilayered physiological response, integrating environmental cues such as diet and microbiota with internal states like metabolism and endocrine regulation to coordinate developmental timing and survival [ 9 ].

Results

Lst is a peptide hormone regulating developmental progression and survival under malnutrition In our recent study [28], we performed transcriptomic profiling of dissected larval midguts from Drosophila reared on a low-yeast diet, a model of malnutrition that elicits developmental adaptation, characterized by reduced larval growth rate and delayed pupariation in wild-type (WT) animals. Given the known role of the microbiome in buffering nutritional stress, we compared germ-free larvae with animals mono-associated with Lactiplantibacillus plantarumWJL (LpWJL, referred as Lp here), a well-characterized member of the Drosophila gut microbiota that recapitulates the growth-promoting effects of a complex microbiome under malnutrition [25]. Among the differentially regulated transcripts, we identified Limostatin (Lst) as significantly upregulated in germ-free larvae raised under malnutrition (Fig 1A) [28]. Lst was previously characterized in adult flies as a peptide hormone secreted by corpora cardiaca during fasting, where it suppresses dIlp production and release from the brain [27]. Our data suggest that Lst may also contribute to the adaptive growth response of germ-free larvae exposed to malnutrition. To test this hypothesis, we analyzed Lst loss-of-function mutants. In line with previous findings, heterozygous and homozygous Lst null mutant (Lst1) animals showed normal developmental progression under standard nutritional conditions [27] (Fig 1B), but a severe developmental lethality was observed in homozygous mutants (Fig 1C). However, under malnourished conditions (low yeast diet, 7 g/L), both heterozygous and homozygous Lst null mutants exhibited significantly accelerated development (Fig 1D). Yet, heterozygous animals displayed reduced viability during development and homozygous animals had high lethality (Fig 1E) relative to their WT siblings and normal nutrition controls. PNG larger image

TIFF original image Download: Fig 1. Lst is a peptide hormone regulating adaptive growth and survival under malnutrition. (A) Transcript levels of gut-derived Lst analyzed via midgut bulk RNA-seq data from germ-free (GF) and Lactiplantibacillus plantarum (Lp)-associated undernourished yw larvae (n = 5). The complete RNA-seq dataset and statistical analyses are available in Bai and colleagues (2026) [28] and deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE310159. (B–E) Median time to pupariation (denoted as days after egg laying, dAEL) (B, D) and percentage of embryo-to-adult lethality (C, E) measured for homozygous and heterozygous Lst null mutants, alongside control larvae. Assessments were performed under normal fed conditions (B, C) or under a 7 g/L low-yeast malnutrition diet (D, E) (n ≥ 7). (F–I) Median time to pupariation (F, H) and percentage of L1-to-adult lethality (G, I) measured across genotypes, including trans-heterozygous Lst mutants, heterozygous mutants carrying a CRIMIC insertion, homozygous Lst null mutants, and control larvae. Assessments were performed under normal fed conditions (F, G) or a 7 g/L low-yeast malnutrition diet (H, I) (n ≥ 7). Statistical significance was determined using a two-tailed unpaired t test (A), a two-way ANOVA with Tukey’s multiple comparisons test (B, D), or a one-way ANOVA with Tukey’s multiple comparisons test (C, E, F–I). Exact P values are indicated on the respective panels. The data underlying the graphs can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003911.g001 To determine whether Lst is specifically required for viability under malnutrition, we adopted a trans-heterozygous strategy by crossing the Lst1 null allele (Lst−/−) with a second allele carrying a CRIMIC insertion within the Lst locus, located at the beginning of the sole intron of the Lst gene (LstCR01853, hereafter referred to as LstCRIMIC). To bypass potential early embryonic lethality, we specifically selected first-instar (L1) larvae of each genotype and monitored larval-to-adult development and viability. Strikingly, upon normal nutrition, both Lst−/− and Lst−/CRIMIC larvae displayed normal developmental timing (Fig 1F) and survival rates (Fig 1G) comparable to controls once they enter the L1 stage after hatching. These results indicate that the reduced viability initially observed upon normal nutrition in Lst−/− animals (Fig 1C) results from an embryonic lethality and/or reduced fertilization/oogenesis efficiency in this specific homozygous context. Importantly, under malnutrition conditions, all mutant genotypes (LstCRIMIC/+ - LstCRIMIC/− - Lst−/−) exhibited significantly accelerated developmental timing (Fig 1H) coupled with an increased lethality (Fig 1I). These findings strengthen our conclusion that Lst is a critical regulator of adaptive larval growth, restraining developmental pace in order to preserve larval viability during nutrient stress. These phenotypes were independently confirmed using two distinct Lst-targeting RNAi lines driven by Lst-GAL4 driver [27] (S1A–S1D Fig). Importantly, despite their accelerated developmental progression, Lst-RNAi animals did not exhibit increased food intake (S1E–S1F Fig), indicating that the enhanced developmental rate is not attributable to elevated feeding activity. Together, these findings establish Lst as a peptide hormone that coordinates developmental progression and survival in response to nutritional stress and microbiome depletion. Its expression under these conditions suggests a role as a critical effector in the endocrine network governing adaptive developmental timing.

Lst expression in EECs is sensitive to amino acid of dietary and microbial origins Our findings indicate that Lst expression in EECs is modulated by fluctuations in both diet and microbiota. While Lst was previously shown to be regulated by dietary sugar levels in adult corpora cardiaca cells [27], it is unclear which nutrients influence its expression in the larval midgut. Notably, conventional inactivated yeast-based (oligidic) diets do not allow for a precise dissection of the nutritional components responsible for Lst regulation, as yeast provides a complex mixture of macro- and micronutrients. Although commonly referred to as “low-protein” diets, yeast-reduced regimens likely affect multiple nutritional inputs beyond amino acids availability. To directly test whether dietary amino acids regulate Lst expression and function, we employed the FLYAA holidic diet (HD) system, a chemically defined nutritional framework previously established for dissecting nutrient-microbiota–host interactions in Drosophila [23,33]. Within this system, we altered total amino acid content while maintaining their relative proportions. Specifically, we used a High Amino Acid Holidic Diet (HAAHD; 21.38 g/L total AAs) and a Low Amino Acid Holidic Diet (LAAHD; 8.55 g/L total AAs), the latter of which has been shown to impair developmental growth [23]. Using these HDs, we found elevated Lst transcripts levels in the midgut (Fig 5A) but not in corpora cardiaca (S5A Fig) under amino acid restriction and microbiota depletion, mirroring our previous observations in germ-free animals fed oligidic low-yeast diets. Functionally, we confirmed that Lst is required for coordinating development under these conditions: germ-free Lst¹ mutants (Fig 5B) and germ-free animals subjected to tissue-specific Lst knockdown in EECs (Figs 5C, 5D, S5B, and S5C), but not in corpora cardiaca cells (S5D–S5G Fig), exhibited altered adaptive maturation under the LAAHD regimen. Together, these findings demonstrate that Lst acts as a key hormonal regulator of adaptive developmental growth in response to dietary amino acid availability and microbiota status. PNG larger image

TIFF original image Download: Fig 5. Lst expression in EECs is sensitive to dietary and microbiota-derived AAs. (A) Relative transcript levels of gut-derived Lst analyzed by qRT-PCR in germ-free (GF) and Lp-associated larvae (Lp) reared under HAAHD and LAAHD (n ≥ 5). Data are presented as mean ± SEM. (B) Median time to pupariation measured for homozygous and heterozygous Lst null mutants, alongside control larvae reared under LAAHD (n ≥ 9). (C, D) Median time to pupariation measured under LAAHD for larvae with Lst knockdown in EECs (EEC > UAS-Lst RNAi) using the KK RNAi line (C) and the TRiP RNAi line (D), each compared against its respective controls (n ≥ 9). (E, F) Relative transcript levels of gut-derived Lst analyzed by qRT-PCR (E) and median time to pupariation (F) of larvae reared in HDs where specific AAs produced by Lp were either supplemented to LAAHD or reduced from HAAHD. n = 6 (E), n ≥ 15 (F). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (A–D) or Welch’s ANOVA with Dunnett’s T3 test (E, F). Exact P values are indicated on the respective panels. The data underlying the graphs can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003911.g005 We next explored whether microbiota-mediated nutrient provisioning contributes to Lst repression. Given the role of amino acid availability in regulating Lst expression, and our prior demonstration that commensal bacteria, including Acetobacter pomorum (Ap) and Lp strains, can supplement amino acids to their juvenile host during malnutrition [23], we hypothesized that Lp represses Lst expression by supplying limiting amino acids. To test this hypothesis, we raised germ-free larvae on either HAAHD or LAAHD that were selectively depleted or supplemented with the specific amino acids that Lp is able to produce (His, Lys, Met, Phe, Thr and Asp. Fig 5E and 5F). Consistent with our model, Lst expression was significantly repressed and developmental progression improved when His, Lys, Met, Phe, Thr and Asp were added to the LAAHD (Fig 5E and 5F). Conversely, reducing the concentration of those same AAs from the HAAHD to their levels in LAAHD led to Lst induction and maturation delay (Fig 5E and 5F). Similarly, association of germ-free larvae raised on LAAHD with Ap, a commensal bacteria capable of producing all amino acids, antagonized Lst expression upon malnutrition (S5H Fig), indicating that microbial production of amino acids may contribute to Lst-dependent developmental growth regulation.

Cellular amino acid sensors are dispensable for Lst induction in EECs We next sought to identify how amino acid restriction leads to Lst induction specifically in EECs. A straightforward hypothesis is that Lst⁺ EECs directly sense amino acid scarcity in the gut lumen through conserved intracellular nutrient-sensing pathways. Two major evolutionarily conserved signaling hubs govern cellular responses to amino acid availability: the Gcn2 kinase, which is activated by uncharged tRNAs and signals amino acid deprivation [34], and the target of rapamycin complex 1 (TORC1), a central regulator of growth and metabolism activated by intracellular amino acids and negatively regulated by the Tsc1/2 complex [35]. To assess the functional requirement for these pathways in Lst⁺ EECs, we used EEC-specific RNAi to knock-down Tsc1 or Gcn2 (Fig 6A, 6B, 6C, and 6D, respectively). In both cases, Lst induction (Fig 6A and 6C) and developmental maturation rates (Fig 6B and 6D) in undernourished germ-free larvae remained unaffected, indicating that neither Gcn2 nor repression of TORC1 signaling in EECs contribute to Lst expression under amino acid-restricted conditions. Together, these results suggest that Lst induction in response to amino acid limitation occurs independently of the canonical intracellular amino acid sensors Gcn2 and TORC1 in EECs. PNG larger image

TIFF original image Download: Fig 6. Cellular amino acid sensors are dispensable for Lst induction in EECs. (A–D) Under malnutrition, relative transcript levels of gut-derived Lst were analyzed by qRT-PCR (A, C) and median time to pupariation was measured (B, D) in germ-free (GF) larvae with EEC-specific Tsc1 knockdown (A, B) or Gcn2 knockdown (C, D). n = 6 for (A, C), n = 11 for (B, D). In (A, C), data are shown as mean ± SEM. Statistical significance was determined using a two-tailed Mann–Whitney U test (A), two-tailed unpaired t test (B–D). Exact P values are indicated on the respective panels. The data underlying the graphs can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003911.g006

Lst induction in EECs is controlled by an inter-organ nutrient sensing regulatory loop The IIS-dependent control of Lst raised the question of how nutrient availability modulated IIS in Lst+ EECs. In Drosophila, the fat body serves as a central nutrient-sensing organ that communicates systemic nutritional status to IPCs in the brain. Circulating amino acid uptake via the transporter slimfast (slif) and activation of the nutrient-responsive TORC1 pathway in the fat body promote the secretion of dIlps, particularly dIlp2, by IPCs [17]. Based on this, we hypothesized that Lst repression in EECs is not simply a local response to nutrient availability but is rather a gut response to low dIlp2 release by IPCs under nutritional stress. To test this, we impaired fat body nutrient sensing by overexpressing Tsc1/2, or by RNAi-mediated knockdown of slif or Raptor (Raptor being a TORC1-specific component [40]) using Lpp-GAL4 [41]. Each manipulation, known to suppress fat body TORC1 signaling, further delayed development under malnutrition (S8A–S8C Fig) and led to modest induction of Lst in the midgut despite the presence of microbial AAs (Fig 8A–8C). These findings suggest that TORC1 activity in the fat body is required to repress Lst expression in EECs in response to systemic cues. Because dIlp2 secretion by IPCs is a direct output of fat body nutrient sensing [17], we next asked whether reduced dIlp2 production in IPCs is sufficient to trigger Lst expression. Pan-neuronal knockdown of dIlp2 using Elav-GAL4 [42] significantly elevated Lst expression in the midgut (Fig 8D) and delayed larval maturation into pupae (Fig 8E), phenocopying the effects of fat body TORC1 inhibition. PNG larger image

TIFF original image Download: Fig 8. Lst is an enterokine involved in an inter-organ regulatory loop impacting circulating levels of dIlp2 in response to nutrient and microbiota status. (A–D) Systemic TORC1 and insulin signaling in the fat body and central nervous system modulate gut Lst expression during malnutrition. Relative transcript levels of gut-derived Lst were analyzed by qRT-PCR in Lp-associated larvae with: (A) Overexpression of Tsc1/2 in the fat body using the Lpp-GAL4 (Lpp > UAS-Tsc1/2), compared to their respective overexpression controls; (B) Knockdown of slif in the fat body (Lpp > UAS-slif RNAi) compared to GD controls. (C) Knockdown of Raptor in the fat body (Lpp > UAS-Raptor RNAi) compared to KK controls; or (D) Knockdown of dIlp2 in neurons including IPCs (Elav>UAS-dIlp2 RNAi), compared to KK controls (n ≥ 5). (E) Median time to pupariation was measured for Lp-associated larvae with neuron-specific dIlp2 knockdown (Elav>UAS-dIlp2 RNAi) and their respective KK controls (n ≥ 12). (F–J) Under malnutrition, tissue responses were characterized in germ-free larvae with EEC-specific Lst knockdown (EEC > UAS-Lst RNAi) and their respective KK controls. (F) Relative transcript levels of dIlp2 in the brain analyzed by qRT-PCR. (G) Representative images of dIlp2 immunostaining in the brain; scale bar: 50 μm. (H) Circulating dIlp2(HF) levels in the hemolymph of heterozygous Ilp2¹ gd2HF larvae measured by ELISA (Genotype: EEC-GAL4/ + ; UAS-Lst RNAi/ + ; Ilp2¹, gd2HF/+). (I, J) Relative transcript levels of InR analyzed by qRT-PCR in the carcass (I) and fat body (J). n ≥ 6 (F), n = 14 (H), n = 5 (I, J). In (A–D, F, I, J), data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired t test (A, C, D), a two-tailed Welch’s t test (B, E) and one-way ANOVA with Tukey’s multiple comparisons test (F, H–G). Exact P values are indicated on the respective panels. The data underlying the graphs can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3003911.g008 Together, these results define a systemic nutrient-sensing loop linking dietary and microbial amino acid availability to Lst expression. In this loop, circulating amino acid are sensed by TORC1 in the fat body to promote dIlp2 release from IPCs, which maintains high IIS activity and suppresses Lst production in EECs. When circulating amino acid levels drop, reduced fat body TORC1 activity decreases dIlp2 release by the IPCs, leading to FoxO activation in Lst ⁺ EECs and induction of Lst. This inter-organ circuit thus connects systemic amino acid sensing to gut endocrine output, coordinating developmental pace with environmental nutrient supply.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003911

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