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

Maintaining nutritional homeostasis requires animals to select and consume specific nutrients to meet their needs. This selection process is driven by appetites for particular nutrients, a phenomenon known as nutrient-specific appetite. Understanding the mechanisms that detect nutritional needs and translate them into feeding decisions remains a significant challenge. While peripheral signals like leptin and various gut hormones, alongside circulating nutrients, influence food intake, the specific hormonal mechanisms governing nutrient-specific appetites are poorly understood. Drosophila, with its conserved gut structure and many evolutionarily conserved gut-derived hormones, serves as a valuable model for investigating gut-brain signaling and its role in feeding decisions. The Drosophila gut, similar to the mammalian gastrointestinal tract, releases various hormones from specialized enteroendocrine cells (EECs). Gut-to-brain signaling transmits crucial information about the nutritional content of the gut, influencing food intake. For instance, in mammals, ghrelin signals hunger while GLP-1 signals satiety, regulating food intake and preventing overconsumption. This study aims to identify and characterize gut-derived signals that specifically regulate appetite for different nutrients in Drosophila, focusing on the role of the gut in adjusting food choice.

Previous research has established that Drosophila, much like mammals, adjusts feeding behavior based on its internal state. The gut, a major endocrine organ, releases hormones from EECs that play a key role in coordinating food intake and metabolism. Gut-to-brain signaling conveys critical information about the nutritional content of the intestine, influencing feeding decisions. While much is known about the gut-hormonal signaling involved in metabolism, less is understood about how the gut communicates the presence or absence of specific nutrients to adjust food choice. This research gap highlights the need to identify and characterize gut-derived signals that regulate appetite for specific nutrients and drive appropriate food choices to maintain nutritional homeostasis.

The researchers employed several methods to investigate the role of gut-derived hormones in regulating feeding behavior in Drosophila. An in vivo RNA interference (RNAi) screen of secreted factors and receptors in adult Drosophila EECs was performed using the *võila*-GAL4 driver to target the RNAi effect to EECs, in combination with ubiquitously expressed temperature-sensitive GAL80 (*Tub*-GAL80^ts). This allowed for adult-stage-specific gene silencing. The researchers focused on analyzing the impact of adult-restricted, EEC-specific gene knockdown on the sugar-water feeding behavior of fed males and females using the fly liquid-food interaction counter (FLIC) system and dye-consumption assays with standard adult fly food. To quantify sugar intake over longer periods, the capillary feeder (CAFÉ) assay was employed. For short-term intake measurements, flies were pre-conditioned by fasting them for 15 hours. The expression of NPF in dissected midguts and central nervous systems (CNS) was measured using quantitative PCR (qPCR) and immunostaining. To ensure specificity, a second driver, NPF::2A::GAL4 (*NPF*>), was used in combination with pan-neuronal RS7C10-GAL80 to suppress neuronal GAL4 activity. Synthetic NPF peptide injections were used to assess the sufficiency of NPF in regulating feeding behavior. The thermosensitive Transient receptor potential A1 (TrpA1) cation channel was expressed in NPF EECs to induce NPF release, enabling manipulation of NPF secretion. Starvation resistance assays were used to indirectly assess energy storage and mobilization. Measurements of triacylglyceride (TAG) and glycogen levels were conducted to evaluate metabolic changes. To assess the animals' preference between different concentrations of sucrose (1% and 10%), both FLIC and CAFÉ assays were used. For yeast preference assays, flies were first protein-deprived by feeding them on sucrose-only medium for 3 days before the experiment. A two-choice dye-based consumption assay was used to measure preference between sugar and protein-rich yeast food. Additionally, the flyPAD system provided another automated method for measuring feeding preferences in a two-choice assay. Finally, immunohistochemistry and confocal imaging were used to examine NPFR expression in various tissues, including the fat body, insulin-producing cells (IPCs), and AKH-producing cells (APCs). Calcium-signaling activity was measured using a luciferase-based CaLexA reporter to investigate the influence of dietary sugar on NPF EECs. In order to evaluate the influence of dietary sugar on the NPFC cells, the researchers also performed RNAi-mediated silencing of Mondo/ChREBP, a sugar-responsive transcription factor, in NPF EECs of mated females.

The study's key findings center on the identification of neuropeptide F (NPF), a gut-derived hormone in Drosophila, as a crucial regulator of sugar appetite and food choice. Specifically: 1. **Gut NPF suppresses sugar intake:** Knockdown of NPF in enteroendocrine cells (EECs) led to a significant increase in sugar consumption in mated females, while conversely, NPF injection suppressed sugar intake. This effect was independent of changes in haemolymph sugar levels, suggesting a direct effect on feeding behavior rather than indirect metabolic effects. 2. **Gut NPF regulates food choice:** The study demonstrated that the loss of gut-derived NPF in mated females not only increased sugar consumption but also significantly reduced their preference for protein-rich food (yeast). Mated females with EEC-specific NPF knockdown showed a reduced preference for yeast, which was not rescued by mating, contrasting with the increased preference for yeast observed in control mated females. 3. **Mating induces NPF secretion:** Mating increased NPF secretion from the midgut, a process dependent on sex peptide (SP) signaling, potentially explaining the shift towards increased protein intake after mating. 4. **NPF acts downstream of SP signaling:** The findings revealed that NPF acts downstream of SP/SPR signaling, mediating the SP-induced increase in protein consumption. Injecting NPF into virgin females increased their preference for yeast, mirroring the post-mating effect. Moreover, the increased preference for yeast following mating was absent in females with SPR knockdown in the Ppk neurons, underscoring SP signaling's critical role in regulating food choice. 5. **Sut2 regulates NPF release:** Knockdown of sugar transporter 2 (*sut2*) in NPF+ EECs increased sugar feeding behavior and intake, mirroring the effects of NPF knockdown. This suggests that Sut2 is involved in regulating NPF production or release. Further research indicates that Mondo/ChREBP-mediated sugar sensing may contribute to NPF regulation in EECs. 6. **NPF suppresses energy mobilization in adipose tissues:** Knockdown of the NPF receptor (NPFR) in the fat body did not replicate the sugar-overconsumption phenotype observed with gut-specific NPF knockdown, but it did lead to decreased sucrose intake and sugar feeding behavior, indicating a metabolic role in energy homeostasis rather than direct regulation of sugar appetite. 7. **NPF regulates food choice through glucagon-like signaling:** The study identified AKH, a glucagon-like hormone, as a key mediator of NPF's effects on feeding behavior. Knockdown of NPFR in AKH-producing cells (APCs) resulted in decreased AKH peptide levels, reduced TAG and glycogen levels, and increased starvation sensitivity. Further studies showed that NPF acts at multiple points to suppress the AKH axis, inhibiting the release of AKH and the AKH-stimulatory factor AstC from midgut EECs. 8. **AKH regulates appetites for sugar and protein-rich food:** The study demonstrated that AKH promotes sugar preference and suppresses protein intake. AKH mutant females exhibited reduced sugar intake and increased yeast preference. Conversely, activation of AKH release increased sugar intake while decreasing yeast intake. Mating suppressed AKH release, which increased yeast consumption. NPF injection replicated this suppressive effect on AKH secretion.

This study significantly advances our understanding of the hormonal mechanisms that regulate nutrient-specific appetites. The identification of NPF as a key satiety signal for sugar and a regulator of protein intake provides a mechanistic basis for how animals match nutrient ingestion to their needs. The finding that NPF acts through an AKH-mediated pathway is novel, highlighting a complex interplay between gut hormones and metabolic regulation. The study also emphasizes the importance of considering sex-specific differences in these mechanisms, as the impact of NPF and AKH on feeding behaviors differs between males and females. The role of Sut2, a sugar transporter, in regulating NPF release adds another layer of complexity to the understanding of glucose sensing and its effects on feeding behavior. The potential of exploiting such appetite-regulatory hormones, such as NPF, for therapeutic interventions in obesity and other metabolic disorders is noteworthy.

This research provides compelling evidence that the gut-derived hormone NPF acts as a key regulator of nutrient-specific appetites in Drosophila. It directly affects sugar satiety and indirectly influences protein consumption through a complex interaction with the AKH pathway. The identified NPF-AKH axis offers a valuable model for understanding how animals adjust nutrient intake to maintain metabolic homeostasis. Future research should focus on elucidating the precise molecular mechanisms underlying NPF release, the interactions between NPF and other gut hormones, and the translational potential of targeting similar pathways in mammals for treating metabolic disorders.

The study primarily focuses on mated female Drosophila, limiting the generalizability of the findings to other sexes and species. The use of RNAi knockdown introduces the possibility of off-target effects, which could influence the results. While the study demonstrates a strong correlation between NPF signaling and feeding behavior, further research is needed to fully elucidate the causal relationships between these factors. The use of indirect measures for energy storage and mobilization may have limitations in accurately reflecting the complete metabolic picture.