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

The ability to sense hunger and satiety is critical for maintaining energy balance. In mammals, hypothalamic neurons, such as those expressing pro-opiomelanocortin and melanocortin-4 receptor, act as satiety sensors, responding to signals from the circulatory system, gastrointestinal tract, and fat depots to regulate food intake. Dysfunction in this system contributes to obesity and metabolic disorders. Drosophila melanogaster, with its conserved metabolic regulatory components, provides a valuable model for studying satiety sensing. Insulin-producing cells (IPCs) in the Drosophila brain are key regulators of feeding and metabolism, indirectly sensing glucose via upstream neurons like those producing adipokinetic hormone (AKH) and those expressing short neuropeptide F (sNPF) and corazonin (Crz). IPCs also respond to other nutrients and fat depot signals. However, the role of IPCs as universal satiety sensors and the interplay of various feeding modulatory cues (NPF, Hugin, DTK, Allatostatin A, drosulfakinin, leukokinin) with IPCs remain unclear. This study aimed to investigate the neural basis of satiety sensing in Drosophila, focusing on neuropeptide signaling.

Existing literature establishes the importance of satiety sensing in maintaining energy balance, highlighting the role of specific neuronal populations in mammals. Research on Drosophila has identified IPCs as central regulators of feeding behavior and metabolism, but their role as primary satiety sensors and the precise mechanisms by which they integrate multiple nutrient signals were not fully understood. Studies have shown that IPCs indirectly sense glucose through AKH-producing cells and glucose-sensing neurons expressing sNPF and Crz, and directly sense branched-chain amino acids and potentially fructose. Additionally, IPCs receive feedback from the fat body regarding fat storage levels. The roles of various neuropeptides in feeding regulation were known, but their precise interactions and contributions to satiety sensing remained an open question.

The researchers employed a multifaceted approach. Initially, a neuron-specific RNAi screen using a manual feeding assay (MAFE) was conducted to identify neuropeptidergic cues involved in satiety and feeding suppression. This assay measured food consumption independently of food-seeking behavior using non-nutritive L-glucose to avoid confounding effects of nutrient sensors. Positive hits from the initial screen were validated using a different pan-neuronal GAL4 driver. The study then investigated the DTK-TAKR99D pathway, generating knock-in GAL4 alleles (DTK^GAL4 and TAKR99D^GAL4) to characterize DTK+ and TAKR99D+ neurons. Acute silencing and activation of DTK neurons were achieved using Shibire (Shi^ts) and dTRPA1, respectively, to assess their impact on food consumption. Quantitative RT-PCR was used to measure DTK mRNA levels in starved and fed flies. D-glucose injections into the fly thorax were performed to assess the impact on feeding behavior. Ex vivo calcium imaging was conducted to identify glucose-responsive DTK neurons. nSyb-GRASP was utilized to examine direct synaptic connections between DTK neurons, TAKR99D neurons, and IPCs. In vivo calcium imaging was performed to observe neuronal activity during feeding. Optogenetics was employed to activate DTK+ and TAKR99D+ neurons and observe downstream effects on IPCs using CsChrimson and GCaMP6m. Finally, the behavioral effects of silencing and activating relevant neuronal populations were assessed using RNAi knockdown and optogenetic manipulation.

The RNAi screen identified DTK-TAKR99D signaling as a potent feeding suppressor. Knockdown of DTK or TAKR99D, or mutations in their respective genes, resulted in increased food consumption. A knock-in GAL4 allele, DTK^GAL4, revealed widespread DTK expression in the fly brain. Acute silencing of DTK neurons increased food consumption, while activation decreased it. DTK expression was reduced by starvation and rapidly induced by re-feeding, suggesting a role in satiety sensing. Injection of D-glucose suppressed feeding, an effect eliminated by silencing DTK neurons. Calcium imaging revealed that only two pairs of DTK neurons in the superior medial protocerebrum (SMP) cluster responded to D-glucose. These SMP DTK+ neurons did not respond to D-fructose or L-glucose, demonstrating specificity. TAKR99D+ neurons in the pars intercerebralis (PI) region were identified as downstream targets of DTK neurons, with direct synaptic connections revealed by nSyb-GRASP. Further, nSyb-GRASP confirmed direct synaptic connections between TAKR99D+ neurons and IPCs. Optogenetic activation of either DTK+ or TAKR99D+ neurons elicited calcium responses in IPCs, confirming functional synaptic transmission. In vivo calcium imaging showed that during feeding, PI TAKR99D+ neurons and IPCs were activated by D-glucose ingestion, with weaker responses to D-fructose and L-glucose. RNAi knockdown of TAKR99D abolished the response of TAKR99D+ neurons and reduced the D-glucose response of IPCs in flies with mutations in *Dtk* and *Takr99d* genes. Silencing TAKR99D+ neurons and IPCs increased food consumption, while activating IPCs reduced it. Overall, a two-synapse circuitry, comprised of SMP DTK+, PI TAKR99D+, and IPCs, functions as a satiety sensor.

This study successfully identified a novel satiety sensing mechanism in Drosophila, demonstrating a two-synapse neural circuitry involving DTK, TAKR99D, and IPCs. The rapid response of this circuitry to changes in circulating glucose during feeding episodes highlights its crucial role in regulating food intake. The specificity of the response to D-glucose suggests a refined mechanism for sensing and responding to this key energy source. The findings provide a significant advance in understanding how nutrient sensing and feeding regulation are integrated within the Drosophila brain. The conserved nature of metabolic pathways between flies and mammals suggests that this research might offer insights into the architecture of satiety sensing mechanisms in mammals and how they are affected by metabolic disorders. The identified circuitry provides a promising model system for future investigations.

This research identified a novel satiety sensor in Drosophila consisting of a two-synapse circuitry involving SMP DTK neurons, PI TAKR99D neurons, and IPCs. This circuitry rapidly responds to increases in circulating D-glucose during feeding, suppressing further food intake and contributing to energy homeostasis. Future studies should investigate the interaction of this circuitry with other satiety sensors and explore potential translational implications for mammalian metabolic diseases.

The study primarily focused on D-glucose sensing; the role of this circuitry in response to other nutrients requires further investigation. The ex vivo calcium imaging experiments showed slower responses compared to in vivo experiments, potentially due to differences in stimulus penetration kinetics. The activation of TAKR99D+ neurons proved lethal, requiring further investigation into the role of other TAKR99D+ neuron clusters.