The survival and well-being of animals rely on precisely maintained balance between energy intake and expenditure. As strict heterotroph, animals need to accurately detect the internal energy state, and continuously search for, occupy, exploit, and consume desirable food sources for adequate and balanced energy intake. Over the past 20 years, there has been extensive research on the regulation of feeding behavior in the field. However, the mechanism of food seeking, a prerequisite of food consumption and a vital component of energy intake, has remained largely unexplored.

The Wang lab established the first quantitative food-seeking assay in fruit flies. They found that starvation induced sustained and reversible locomotor activity in adult fruit flies, and that starvation-induced hyperactivity was directed toward the localization and acquisition of food sources, because it could be suppressed upon the detection of food cues via both central nutrient-sensing and peripheral sweet-sensing mechanisms, via induction of food ingestion. By using this quantitative behavioral assay, they screened major neurotransmitter and neuropeptide molecules and found that octopamine (OA), the insect counterpart of vertebrate norepinephrine, as well as the neurons expressing OA, were both necessary and sufficient for starvation-induced food seeking. OA was not required for starvation-induced changes in food consumption, suggesting independent regulations of energy intake behaviors upon starvation. Taken together, their results established a quantitative behavioral paradigm to investigate the regulation of food-seeking behavior and identified a conserved neural substrate that linked organismal metabolic state to a specific behavioral output.

They also identified the core neural circuitry underlying food-seeking behavior. The establishment of a quantitative food-seeking assay and the identification of OA as a key neural regulator paved the way for further investigation of the regulatory mechanism of food-seeking behavior. By conducting a systematic RNAi screen using their food-seeking assay, they identified the receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, required for starvation-induced food seeking. Importantly, they also found that AKHR was expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR⁺ neurons and blocking OA signaling in these neurons both eliminated starvation-induced food seeking, whereas activation of these neurons accelerated the onset of food seeking upon starvation. Neither AKHR nor these AKHR⁺OA⁺ neurons were involved in increased feeding upon starvation, again suggesting that starvation-induced food seeking and feeding were independently regulated. Moreover, single cell RNAseq of AKHR⁺OA⁺ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposed suppressive effect on starvation-induced food seeking. Therefore, insulin and glucagon signaling exerted opposite effects on starvation-induced food seeking via a common OA target in Drosophila.

They elucidated how unhealthy diet disrupted the regulation of normal food-seeking behavior. Similar to mammalian studies, they found that high-fat diet (HFD) greatly enhanced starvation-induced food seeking without interfering with flies’ metabolism as well as their feeding behavior. Such an effect might contribute to the further enhancement of food intake and the development of obesity upon HFD feeding. They found that HFD enhanced the excitability of the core neural circuitry discussed in previous sessions (AKHR⁺OA⁺ neurons), to the hunger hormone AKH, via increasing the accumulation of its cognate receptor AKHR in these neurons. Therefore, the same group of OA neurons that regulate starvation-induced food seeking was also the neural target affected by HFD.

Another major scheme of the lab is to study how the CNS detects fluctuating levels of vital nutrients, especially sugars and amino acids, and how it triggers different behavioral output accordingly to maintain the homeostasis of these nutrients. Rodent studies have highlighted a central role of the neurons located in the hypothalamus in sensing internal energy state and regulating food intake, including those expressing orexigenic peptides neuropeptide Y and agouti related protein, and those expressing anorexigenic precursor pro-opiomelanocortin. However, recent studies have challenged the requirement of these neurons in feeding regulation, highlighting the complexity of how the brain senses and responds to different types of nutrients at the cellular and molecular levels and the need to systematically identify novel nutrient sensors.

To this aim, Wang lab developed a simple yet quantitative food consumption assay, named Drosophila Manual Feeding (MAFE) assay, in which individual adult flies were immobilized and presented directly with fine capillary filled with liquid food and their food consumption was measured for each feeding episode. By using this assay, they conducted a systematic RNAi screen and identified Drosophila Tachykinin (TK), the fly homolog of mammalian substate P, and its cognate receptor TAKR99D, as potent feeding suppressors (and putative nutrient sensors). Indeed, although TK was expressed in a number of neuronal clusters in the fly brain, they found that only two pairs of TK⁺ neurons in the superior medial protocerebrum region could be directly activated by elevated levels of circulating D-glucose and imposed a suppressive effect on feeding. Moreover, these TK⁺ neurons formed a two-synapse circuitry targeting insulin-producing cells, a well-known feeding suppressor, via TAKR99D⁺ neurons, and this circuitry could be rapidly activated during food ingestion and cease feeding. Taken together, they identified a novel sugar sensor in the fly brain that could detect specific circulating nutrients and in turn modulate feeding, shedding light on the neural regulation of energy homeostasis.

Besides sugar sensors, Wang lab has been very interested in understanding how the CNS detects amino acids. As vital nutrients, amino acids could be detected by the peripheral nervous system of various animal species to trigger an appetitive “umami” taste. mTOR signaling in the hypothalamus also played a role in sensing circulating amino acids and suppressing feeding in rodent models. However, it remained largely unclear how the brain detects the composition of amino acids in the diet and elicits differential responses. In the past years, Wang lab has identified two central amino acid sensors with distinct functions in metabolism and behavior.

They identified a post-ingestive amino acid sensor in the fly brain. By using the MAFE assay, they found that three amino acids, L-glutamate, L-alanine, and L-aspartate, but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promoted food consumption in the fruit fly. In vivo and ex vivo calcium imaging showed that six brain neurons expressing diuretic hormone 44 (DH44), the fly homolog of mammalian corticotropin-releasing hormone (CRH), could be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44⁺ neurons abolished the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons was sufficient to promote feeding, suggesting that DH44⁺ neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining revealed that a putative amino acid transporter, CG13248, was enriched in DH44⁺ neurons. Knocking down CG13248 expression in DH44⁺ neurons blocked the increase in food consumption and eliminated calcium responses induced by dietary amino acids. Therefore, these data identify DH44⁺ neuron as a key sensor to detect amino acids in a post-ingestive manner and to enhance food intake via a putative transporter CG13248.

They have also identified a specific cysteine sensor in the fly brain that imposed both feeding-suppression and fat-burning effects. Similar to observations from human practice, they found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine directly activated a small group of neurons expressing a neuropeptide FMRFamide (FMRFa) and promoted the release of FMRFa[28]. Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. They also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide[36]. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without behavioral abnormalities. Therefore, their study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases.