A novel satiety sensor detects circulating glucose and suppresses food consumption via insulin-producing cells in Drosophila

Satiety sensing ensures balanced intake of energy and nutrients. In mammals, hypothalamic neuron populations (e.g., POMC- and MC4R-expressing neurons) act as satiety sensors by integrating circulating nutrients, gastrointestinal signals, and fat stores to suppress feeding. Drosophila provides a tractable model with conserved metabolic regulators; insulin-producing cells (IPCs) in the fly brain are established suppressors of feeding and regulators of metabolism. IPCs likely cannot directly sense circulating glucose (lack of KATP), instead receiving inputs from AKH cells and glucose-sensing sNPF/Crz neurons, and can detect other nutrients (branched-chain amino acids autonomously; fructose via Gr43a neurons). IPCs also monitor fat-body-derived signals (upd2, FIT, Stunted). However, whether IPCs constitute the universal satiety sensor and how multiple neuromodulatory cues interact with IPCs to regulate feeding remain unclear. This study systematically probes neuropeptidergic mechanisms of satiety sensing, hypothesizing that specific peptide-receptor circuits detect internal nutrient state to rapidly suppress food intake. An RNAi screen identifies DTK-TAKR99D signaling as a candidate satiety pathway, leading to the discovery of a DTK→TAKR99D→IPC circuit that senses circulating D-glucose during meals to terminate feeding.

Prior work has delineated central satiety and nutrient sensing across species. In rodents, POMC and MC4R circuits suppress feeding by integrating hormonal and nutrient signals. In Drosophila, IPCs regulate feeding and metabolism but likely do not directly sense blood glucose due to absent KATP channels; they are influenced by upstream AKH cells and brain glucose-sensing neurons co-expressing sNPF and corazonin. IPCs can directly sense branched-chain amino acids (via LAT1-like transport) and receive fructose-sensitive input from Gr43a-expressing neurons. Fat-body-derived signals (upd2/leptin-like, FIT, Stunted) modulate IPC activity, linking adiposity to satiety. Several neuropeptides modulate feeding (NPF, Hugin, DTK, Allatostatin A, DSK, leukokinin), suggesting a complex neuromodulatory network. Prior reports indicated DTK receptor expression in IPCs, and tachykinin signaling has roles in metabolism and behavior, but a defined circuit for glucose-driven satiety signaling had not been established.

• Genetic screening and lines: A neuron-specific RNAi screen targeting neuropeptide receptors was conducted using pan-neuronal drivers (elav-GAL4, nSyb-GAL4). UAS-RNAi lines and various transgenic tools were sourced primarily from BDSC. Knock-in GAL4 alleles were generated: DTKGAL4 (GAL4 fused to DTK via T2A at the endogenous locus) and TAKR99DGAL4 (GAL4 inserted into TAKR99D exon 1) using CRISPR/Cas9-mediated homologous recombination.
• Feeding assays: The MAFE (Manual Feeding) assay quantified single-meal food intake by presenting 500 mM L-glucose (non-nutritive) or D-glucose via capillary to immobilized flies; consumption volume was measured per individual. Dye-labeled group feeding (FD&C Blue #1) measured 1 h intake in groups of 10. Proboscis extension reflex (PER) assessed feeding initiation in response to sucrose.
• Manipulations of neuronal activity: Acute silencing via temperature-sensitive Shibirets at 30°C; activation via dTRPA1 at 30°C. Optogenetic activation used UAS-CsChrimson with all-trans-retinal supplementation and red LED stimulation in ex vivo brain prep.
• Hemolymph nutrient manipulation: Thoracic injection (~50 nL) of AHL buffer with or without 100 mM D-glucose to elevate circulating glucose before MAFE assays.
• Imaging and connectomics: Ex vivo calcium imaging (GCaMP6m) of DTK+, TAKR99D+, and IPCs in dissected brains perfused with sugars (typically 80 mM D-glucose), recording ΔF/F over time across defined DTK neuron clusters (AL, SEZ, SLP, SMP, VMNP, INP). In vivo calcium imaging involved exposing the dorsal brain of intact flies; feeding episodes were delivered to the proboscis while recording neuronal activity at 1 fps. Synaptic connectivity was assessed by nSyb-GRASP between DTK+ and TAKR99D+ neurons, and between TAKR99D+ neurons and IPCs; controls lacked one GRASP component. Photoactivatable GFP (C3PA-GFP) traced SMP DTK neuron projections to PI. Optogenetic activation of DTK+ or TAKR99D+ neurons while recording IPC calcium responses tested functional synapses.
• Behavioral controls: Two-choice feeding preference assays contrasted nutritive D-glucose vs non-nutritive L-glucose. Locomotion was monitored with DAMS to assess confounds.
• Molecular assays: qRT-PCR measured DTK mRNA in heads and bodies of fed, 24 h starved, and re-fed flies to assess nutritional regulation of DTK expression.
• Statistics: Normality checked by D'Agostino-Pearson. Student's t-test for pairwise comparisons; one-way ANOVA with Bonferroni post hoc for multiple groups; Dunnett's post hoc for screening hits. Sample sizes varied by assay (e.g., n≈15–35 in MAFE; imaging cohorts n≈6–16).

• DTK-TAKR99D as feeding suppressors: In a pan-neuronal neuropeptide receptor RNAi screen (MAFE with 500 mM L-glucose), TAKR99D knockdown uniquely and reproducibly increased food intake among positive hits. Global RNAi knockdown and genetic mutants of Dtk and Takr99d significantly increased L-glucose consumption; mutants also overconsumed nutritive D-glucose, indicating DTK-TAKR99D signaling suppresses feeding without affecting starvation-induced food seeking, body weight, or length.
• DTK neuron function: Acute silencing of DTK+ neurons (Shits at 30°C) increased intake of both L- and D-glucose. Activation of DTK+ neurons (dTRPA1 at 30°C) reduced PER to sucrose and decreased long-term food consumption in group assays, confirming a suppressive role.
• Nutritional regulation and satiety signaling: DTK mRNA decreased with 24 h starvation (heads and bodies) and was rapidly restored upon re-feeding, supporting DTK involvement in satiety state. Elevating hemolymph D-glucose by thoracic injection (100 mM in AHL) significantly reduced subsequent food intake; this effect required DTK+ neuron activity, as silencing DTK neurons abolished the suppression.
• Identification of glucose-sensing DTK neurons: Among six DTK+ brain clusters, only the SMP cluster showed robust calcium responses to D-glucose in ex vivo preparations. At single-cell resolution, just two pairs of SMP DTK+ neurons (one per hemisphere) exhibited strong oscillatory activation to D-glucose, but not to D-fructose or L-glucose, indicating specificity for circulating D-glucose.
• Circuit architecture: nSyb-GRASP revealed synaptic contacts from DTK+ to TAKR99D+ neurons in the antennal lobe, pars intercerebralis (PI), and fan-shaped body. Six TAKR99D+ neurons were localized in the PI near but non-overlapping with IPCs. GRASP demonstrated direct synapses between TAKR99D+ neurons and IPCs; no direct DTK→IPC synapses were detected, establishing a two-synapse DTK (SMP) → TAKR99D (PI) → IPC circuit.
• Functional connectivity: Optogenetic activation of DTK+ or TAKR99D+ neurons elicited robust calcium increases in IPCs; controls without CsChrimson showed no responses, confirming functional synaptic transmission.
• Rapid activation during feeding: In vivo calcium imaging showed that ingestion of D-glucose rapidly activated PI TAKR99D+ neurons and IPCs during feeding episodes (seconds timescale). Responses to D-fructose and L-glucose were significantly weaker in TAKR99D+ neurons and IPCs; IPCs showed some fructose responsiveness likely via Gr43a inputs and a very weak but significant response to L-glucose via unknown mechanisms.
• Genetic dependence: RNAi knockdown of TAKR99D in TAKR99D+ neurons abolished their D-glucose feeding-evoked responses. IPC responses to D-glucose were markedly reduced in Dtk and Takr99d mutants, demonstrating requirement of DTK-TAKR99D signaling.
• Behavioral outcomes of circuit manipulation: Silencing TAKR99D+ neurons or IPCs increased L-glucose intake, mirroring DTK neuron silencing; activating IPCs reduced group food consumption. Activation of TAKR99D+ neurons was lethal (suggesting essential functions of some TAKR99D+ populations).
• Model: Two pairs of SMP DTK+ neurons act as internal D-glucose sensors; upon nutrient ingestion and hemolymph glucose rise, DTK release activates PI TAKR99D+ neurons, which in turn activate IPCs to cease feeding, preventing overconsumption and supporting energy homeostasis.

The study addresses the central question of how satiety is sensed and relayed to suppress feeding in Drosophila. It identifies a discrete neuropeptidergic pathway in which two pairs of SMP DTK+ neurons detect rises in circulating D-glucose and transmit this satiety signal via TAKR99D+ neurons in the PI to activate IPCs, known suppressors of feeding. This mechanism operates on a seconds timescale, matching the short duration of fly meals and enabling rapid termination of ingestion. The specificity for D-glucose distinguishes this internal satiety sensor from fructose-sensitive Gr43a pathways and from amino acid or adiposity signals, highlighting parallel nutrient-specific channels converging on IPCs. The synaptic mapping and functional manipulations (optogenetics, GRASP, genetic disruptions) provide convergent evidence that the DTK→TAKR99D→IPC circuit is necessary and sufficient to modulate feeding suppression in response to internal glucose. These findings integrate with broader models in which IPCs function as a satiety hub, receiving diverse nutrient and adiposity inputs to orchestrate feeding and metabolic homeostasis.

This work establishes a novel, rapid satiety-sensing circuit in Drosophila composed of two pairs of SMP DTK+ neurons, six PI TAKR99D+ neurons, and IPCs. The circuit detects increases in circulating D-glucose during feeding and suppresses further intake, thereby contributing to energy homeostasis. Key contributions include: (1) identification of DTK-TAKR99D signaling as a potent feeding suppressor; (2) discovery of DTK neurons that specifically sense internal D-glucose; (3) mapping and functional validation of a two-synapse DTK→TAKR99D→IPC pathway that is rapidly engaged during meals. Future directions include delineating the dynamics of circuit activation and deactivation during natural feeding bouts, clarifying the roles of DTK/TAKR99D innervation to other regions (e.g., fan-shaped body), resolving mechanisms underlying IPC responses to non-glucose sugars, and integrating this pathway with other satiety and sugar-sensing circuits to build a comprehensive model of nutrient-specific satiety control. Given conserved metabolic principles, these insights may inform understanding of satiety sensing and its dysregulation in mammals.

• The functional role of DTK and TAKR99D projections to the fan-shaped body was not determined.
• Activation of TAKR99D+ neurons was lethal, limiting causal testing and suggesting essential functions in other TAKR99D+ populations that were not dissected.
• IPCs exhibited weak responses to L-glucose and were activated during fructose feeding via other inputs; the mechanisms and integration with the DTK-TAKR99D pathway remain unresolved.
• Ex vivo calcium responses were slower than in vivo, reflecting limitations of stimulus penetration or preparation that may affect quantitative kinetics.
• While the study demonstrates rapid D-glucose sensing and feeding suppression, it does not test long-term metabolic outcomes or generalizability across sexes or life stages beyond adult females.
• The work focuses on D-glucose; interactions with other satiety pathways (e.g., AKH, Gr43a, amino acid sensors, adiposity signals) and how they converge temporally onto IPCs require further investigation.