While a balanced intake of macronutrients — carbohydrates, fats, and proteins — is essential for metabolic homeostasis, animals need higher protein intake during critical life stages like pregnancy. A recent paper in Cell by Wu et al. introduces the novel concept of adjusting protein intake setpoints based on sex and mating status, using two opposing G protein-coupled receptor (GPCR) signaling pathways that regulate protein appetite-controlling neurons in the fruit fly, Drosophila melanogaster.

Animals have evolved nutrient-specific hunger mechanisms to address internal macronutrient imbalances, thereby enhancing their survival. Among macronutrients, protein is essential for growth and development, particularly during maturation and reproduction. For instance, pregnant animals show a strong preference for protein-rich foods due to the need for extra amino acids for offspring development. In fruit flies, mated females have a higher protein intake setpoint and consume more protein to feel satiated compared to virgin females or males, as these amino acids are crucial for egg production.1 However, the precise mechanisms by which animals adjust their protein intake setpoints based on physiological needs, such as pregnancy, remain unclear.

In a recent Cell issue, Wu and colleagues revealed the molecular mechanism behind intrinsic protein intake setpoints based on sex and mating status.2 They developed a novel ‘lid-feeding’ assay to measure daily two-choice food intake (protein or carbohydrate) in Drosophila without food deprivation. Their findings showed that the protein intake setpoint is highest in mated females, followed by virgin females and then males, while carbohydrate intake remains similar across all groups. To understand how sex and mating status affect this setpoint, they measured the membrane potentials of DA-WED neurons regulating protein hunger.1 They discovered that resting membrane potentials are highest in mated females, followed by virgin females and then males, mirroring the differential protein intake among them. Importantly, protein starvation increased action potential frequency without affecting the resting membrane potential. To determine whether differences in resting membrane potentials are responsible for variations in protein intake setpoints, they manipulated the resting membrane potentials of DA-WED neurons by modulating the expression of ORK1, a membrane two-pore domain leaky potassium channel. ORK1 knockdown in males and virgin females depolarized the resting membrane potentials, resulting in higher membrane potentials and increased daily protein intake, demonstrating a causal relationship between the resting membrane potentials of DA-WED neurons and protein intake setpoints. A G protein-coupled receptor (GPCR) screening revealed that two GPCR signaling pathways control the resting membrane potential of DA-WED neurons by modulating the opening and closing of ORK1 channels. Activation of the FMRFamide neuropeptide (FMRFa), its receptor (FMRFaR), and its downstream protein kinase C (PKC) pathway leads to the opening of ORK1, whereas activation of the myosuppressin peptide (MS), its receptor (MSR2), and its downstream protein kinase A (PKA) pathway leads to the closing of ORK1 (Fig. 1). In males, activation of FMRFaR and inactivation of MSR2 lead to the activation of both PKC and PKA, which increases ORK1 channel opening, resulting in a low resting membrane potential and low protein intake setpoint. In virgin females, inactivation of both FMRFaR and MSR2 leads to the inactivation of PKC and activation of PKA, decreasing ORK1 channel opening and resulting in a higher resting membrane potential and a higher protein intake setpoint compared to males. After mating, inactivation of FMRFaR and activation of MSR2 induce the inactivation of both PKC and PKA, leading to the closing of ORK1 channels, which results in the highest resting membrane potential and the highest protein intake setpoint (Fig. 1). Additionally, they found that FMRFa-expressing neurons (FMRFa-WED) and MS-expressing neurons (MS-WED) directly interact with DA-WED neurons, forming an upstream circuit that regulates protein intake (Fig. 1).

Mated females, virgin females, and males activate different neural circuits for DA-WED neuron activation, affecting GPCR signaling and modulating ORK1 channel activity. Changes in ORK1 activity influence resting membrane potential of DA-WED neurons and protein intake setpoints. See the text for more details.

We can anticipate potential upstream signals specific to sex and mating status that modulate FMRFa and MS signaling, regulating the electrophysiological setpoints of DA-WED neurons. Previous studies have shown that the sex peptide (SP) from male seminal fluid activates sex peptide receptor (SPR) signaling in females, leading to post-mating behavioral changes like rejecting further mating and reducing daytime sleep.3,4,5 Additionally, mated females consume more protein than virgin females,1 suggesting that the SP-SPR circuit is crucial for driving physiological changes post mating. Based on these findings, one could speculate that upstream SP-SPR signaling activates MS-WED neurons, which in turn activates MS-MSR2 signaling for PKA inactivation in DA-WED neurons, elevating their resting membrane potential compared to virgin females (Fig. 1). Sexual dimorphism influences various innate behaviors between males and females, including feeding, sleep cycles, aggression, and mating, which are largely regulated by the fruitless (fru)/doublesex (dsx) gene-related circuits and their downstream signaling pathways.6,7 Therefore, it would be interesting to test whether the fru/dsx-related circuit acts upstream of male-specific activation of FMRFa-WED neurons, thereby activating FMRFa-FMRFaR-PKC signaling in DA-WED neurons to lower the protein intake setpoint (Fig. 1). Future research will unravel these upstream signaling mechanisms, particularly the involvement of post-mating signaling and male-specific signaling in the activation of MS-WED and FMRFa-WED neurons, respectively.

This study introduces key advances in nutrient-specific feeding and innate behavior research. It shifts focus from mechanism of starvation-driven homeostatic needs1,8 to how animals adjust nutrient target levels based on physiological demands influenced by sex and mating status. Protein intake setpoints are encoded in hunger neurons as resting membrane potentials, with compensatory feeding behaviors linked to action potential frequency. This research demonstrates that these setpoints can be reprogrammed by altering GPCR signaling in hunger neurons. Since deregulated protein intake setpoints may lead to harmful metabolic and physiological effects, modulating these setpoints may offer a novel therapeutic approach. Additionally, this study paves the way for exploring how intrinsic setpoints for behaviors like sleep, mating, and aggression are influenced by an animal’s development and physiology. In humans, setpoints regulate functions such as eating, emotions, and stress, with imbalances linked to obesity, mood disorders, and sleep issues. This study provides a critical foundation for future research on disorders related to setpoint regulation.