A gut-derived hormone suppresses sugar appetite and regulates food choice in Drosophila

The study addresses how internal nutritional needs are sensed and translated into nutrient-specific appetites that guide food choice. While peripheral hormonal signals such as leptin and various gut hormones are known to influence overall food intake, the mechanisms governing specific appetites (for sugar vs protein) are unclear. Drosophila provides a conserved model in which the gut, a major endocrine organ, communicates nutritional state to the brain to regulate feeding. The authors hypothesize that gut-derived hormonal signals mediate nutrient-specific appetites, particularly that an enteroendocrine hormone signals sugar satiety to reduce sugar intake and promote protein consumption, especially in mated females whose protein needs increase for egg production.

Prior work shows that nutrient-specific appetite exists across species and that gut-to-brain signalling regulates feeding. In mammals, ghrelin promotes hunger, while GLP-1 acts as a satiety signal. In Drosophila, the gut releases conserved hormones from enteroendocrine cells (EECs) that affect feeding and metabolism. Although gut hormonal control of metabolism is increasingly understood, gut signals regulating nutrient-specific appetite had not been defined. Sex peptide (SP) signalling in mated females increases yeast (protein) preference, and recent studies implicated gut hormones such as Allatostatin C (AstC), Bursicon, and NPF in metabolic control and AKH modulation. NPF is orthologous to mammalian NPY-family peptides (including PYY), suggesting potential conserved roles in satiety and nutrient selection.

• Genetic tools and fly stocks: Adult female Drosophila were reared under standard conditions. Tissue-specific RNA interference (RNAi) and GAL4/UAS systems with temperature-sensitive GAL80ts enabled adult-restricted knockdowns. Drivers included voilà-GAL4 for EECs (EEC>), NPF::2A::GAL4 with pan-neuronal GAL80 to target gut NPF cells (NPF>), AKH-GAL4 for AKH-producing cells (APCs), Cg-GAL4 for fat body, AstC::2A::GAL4 for AstC+ EECs, elav-GAL4 for neurons, da-GAL4 for ubiquitous expression, and DILP2-GAL4 for IPCs. NPFR::T2A::GAL4 reporter was used to visualize NPFR expression. TrpA1 was used for thermogenetic activation. CRISPR-based reporters and RNAi lines targeted NPF, NPFR, AKH, sut2, sut1, Mondo/ChREBP, and SPR.
• RNAi screening: An in vivo EEC-specific RNAi screen of secreted factors/receptors assessed sugar-water feeding using the FLIC system in fed males and females.
• Feeding assays: Short-term consumption quantified by dye-based spectrophotometric assay (30 min, erioglaucine/amaranth dyes); long-term intake and sugar preference quantified by CAFÉ capillary assay (6–24 h, 1% vs 10% sucrose choice); automated behaviour monitoring by FLIC (20–24 h feeding interactions) and flyPAD two-choice assays (1 h). Two-choice dye assays measured preference between 10% sucrose vs 10% yeast; yeast preference was also assessed after 3-day protein deprivation on sucrose-only medium. Video-based tracking quantified time spent on yeast vs sugar patches.
• Hormone and peptide manipulations: Synthetic amidated NPF peptide was injected (final approx. 1.25 µM haemolymph concentration). TrpA1-mediated activation of specific cells at 29 °C was used to induce peptide release (NPF or AKH) and behavioural effects, with genetic epistasis via simultaneous knockdown (e.g., AKH RNAi) to confirm mediation.
• Molecular and imaging analyses: qPCR measured transcripts (NPF, AstC, sut2, etc.) in dissected tissues (midgut, CNS, anterior gut). Immunohistochemistry and confocal microscopy quantified peptide levels (NPF in midgut EECs, AKH in APCs, AstC in EECs) and NPFR>GFP expression in tissues (fat body, APCs, AstC+ EECs). CaLexA (LexA::NFAT::VP16 reporters with luciferase or GFP) reported calcium activity history of NPF+ or AstC+ EECs and APCs under starved/refed and mating conditions.
• Nutrient manipulations: Starvation on agar (15–24 h), refeeding with sucrose (2–6 h), 3-day protein deprivation (sucrose-only). Mating manipulations included mating with SP+ or SP-deficient (SP0/Df) males; neuronal SPR knockdown in ppk+ neurons tested SP pathway dependence.
• Metabolic assays: Whole-body triacylglyceride (TAG) and glycogen assays; haemolymph glucose measurements. Starvation-survival assays assessed energy mobilization.
• Statistical analysis: Data normality assessed; appropriate tests applied (Student’s t-test, Mann–Whitney U, one-way ANOVA with Dunnett’s/Tukey’s, Kruskal–Wallis with Dunn’s, Kaplan–Meier/Gehan–Breslow for survival).

• Gut NPF suppresses sugar intake and promotes satiety in females:
  • EEC-specific NPF knockdown increased sugar feeding in mated females across assays (FLIC: increased feeding time; dye and CAFÉ assays: increased intake over 30 min to 6 h; e.g., CAFÉ 6 h sugar intake, P = 0.0005) and increased consumption of mixed sugar+yeast food, indicating hyperphagia. Males showed decreased 24 h food consumption upon gut NPF loss.
  • NPF injection suppressed hyperphagia induced by gut NPF knockdown (dye assay, P = 0.0054 vs control; P = 0.0002 vs NPF-RNAi without injection) without altering haemolymph glucose.
  • Thermogenetic activation of NPF+ EECs via TrpA1 decreased intake; effect abolished by concurrent NPF knockdown.
  • EEC-specific NPF knockdown reduced starvation resistance and lowered TAG and glycogen in females, indicating increased energy mobilization; males’ TAG less affected.
• NPF regulates nutrient-specific food choice, especially after mating:
  • EEC NPF knockdown increased preference for higher sucrose (10% vs 1%) across FLIC and CAFÉ (e.g., preference P = 0.0031–0.0165), supporting a sugar satiety role.
  • Mating reduced midgut NPF peptide levels (consistent with release) and increased NPF EEC CaLexA activity in an SP-dependent manner; mating increased yeast preference in controls but not in EEC NPF knockdown females. NPF injection increased yeast preference in virgins.
  • Females mated to SP+ males showed high yeast preference; EEC NPF knockdown or mating to SP− males reduced yeast preference comparably. NPF injection rescued yeast preference in SP pathway-compromised conditions, placing NPF downstream of SP/SPR signalling.
• Sugar sensing in NPF+ EECs requires Sut2 and involves Mondo/ChREBP:
  • Knockdown of sut2 (SLC2-family transporter) in NPF+ EECs increased sugar feeding and reduced yeast intake and preference, phenocopying NPF loss. sut2 loss caused intracellular NPF peptide accumulation with reduced transcript, indicating secretion defects and NPF retention; sut2 expression was enriched in NPF+ EECs.
  • Starvation decreased CaLexA activity and increased NPF peptide retention in NPF EECs; sucrose refeeding rapidly increased CaLexA (within 2 h) and reduced NPF peptide (by 6 h) without changing NPF mRNA, consistent with secretion.
  • Manipulating Mondo/ChREBP in EECs modestly increased sugar intake when silenced; restoring Mondo after transient knockdown decreased NPF peptide levels (indicative of secretion) without altering transcripts, mimicking post-meal sugar sensing.
• NPF acts on multiple targets to coordinate metabolism and feeding:
  • NPFR expression observed in fat body and APCs; fat-body NPFR knockdown increased overall intake and reduced TAG/glycogen with increased starvation sensitivity, but did not increase sugar preference—indicating a metabolic, not preference, role.
  • Neuronal NPFR knockdown decreased feeding (consistent with central NPF promoting feeding); ubiquitous NPFR knockdown gave intermediate effects.
• NPF suppresses AKH signalling to regulate sugar vs protein appetite:
  • NPFR knockdown in APCs reduced AKH peptide levels in fed state (indicative of increased release), reduced TAG/glycogen, and decreased starvation survival—consistent with elevated AKH signalling.
  • NPFR knockdown in AstC+ EECs increased AstC cell activity (CaLexA) and reduced detectable AstC peptide-positive cells, suggesting enhanced AstC release that promotes AKH; these flies had reduced starvation survival.
  • APC-specific NPFR knockdown increased sugar-directed feeding behaviour, sugar intake, and preference for higher sucrose; it reduced yeast preference in mated females, phenocopying gut NPF loss. AKH knockdown fully rescued the sugar overconsumption phenotype from NPFR knockdown in APCs, indicating AKH mediates NPFR effects.
• AKH biases feeding towards sugar and away from protein:
  • AKH mutants or APC-specific AKH knockdown decreased sugar intake and strongly increased yeast preference and consumption in mated females; activation of APCs (TrpA1) increased sugar intake and decreased yeast intake, effects blocked by AKH knockdown.
  • Sex-specificity: In males, AKH loss decreased yeast intake, indicating dimorphic roles.
  • Mating increased AKH peptide retention and reduced APC CaLexA activity in an SP-dependent manner; NPF injection in virgins increased AKH peptide levels and reduced APC activity, indicating NPF represses AKH secretion. NPF failed to increase yeast preference when NPFR was knocked down in APCs or in AKH mutants, placing AKH downstream of NPF. Overall, in mated females, sugar ingestion activates Sut2/Mondo-dependent NPF release from gut EECs, which suppresses AKH signalling (directly in APCs and indirectly via AstC EECs) and antagonizes AKH action in fat body, thus reducing sugar appetite and promoting protein-rich yeast consumption.

The findings demonstrate a gut-brain-adipose hormonal axis that translates nutrient detection into nutrient-specific appetites. Gut-derived NPF acts as a sugar-induced satiety hormone in mated female Drosophila, shifting feeding away from sugar and towards protein. Mechanistically, NPF release from EECs requires sugar sensing via Sut2 and involves Mondo/ChREBP, and NPF signals through NPFR on APCs to repress AKH release. AKH, traditionally a generic hunger hormone, is shown here to specifically promote sugar appetite and suppress protein intake. NPF also modulates metabolism by acting on fat body NPFR to promote energy storage and oppose AKH-driven mobilization. This axis operates downstream of mating-induced SP/SPR signalling, thereby explaining mating-associated increases in protein appetite. The work aligns Drosophila NPF with mammalian NPY-family gut peptides (notably PYY) and suggests conserved principles whereby gut hormones mediate macronutrient-specific satiety and food choice.

This study identifies gut-derived NPF as a key sugar satiety hormone that suppresses sugar appetite and promotes protein intake in mated female Drosophila by repressing AKH signalling and modulating adipose metabolism. It delineates a multi-level NPF–AKH axis (gut EECs → APCs → fat body) integrating nutrient sensing (via Sut2 and Mondo/ChREBP) with behavioural choice. The work reveals a sex- and mating-dependent regulatory circuit for nutrient-specific appetite and reframes AKH as a driver of sugar-specific hunger. Given NPF’s homology to mammalian NPY-family peptides and Sut2’s homology to human GLUT7, these findings point to conserved mechanisms and potential therapeutic targets to modulate nutrient-specific appetites. Future research should examine whether mammalian intestinal GLUT7 influences gut hormone secretion and whether human PYY or related pathways specifically regulate sugar vs protein appetite.