Identification of a neural pathway governing satiety in Drosophila

Abstract

Satiety cues a feeding animal to cease further ingestion of food, thus protecting it from excessive energy gain. Impaired control of satiety is often associated with feeding-related disorders such as obesity. In our recent study, we reported the identification of a neural pathway that expresses the myoinhibitory peptide (MIP), critical for satiety responses in Drosophila. Targeted silencing of MIP neuron activity strikingly increased the body weight (BW) through elevated food intake. Similarly, genetic disruption of the gene encoding MIP also elevated feeding and BW. Suppressing the MIP pathway behaviorally transformed the satiated flies to feed similar to the starved ones, with augmented sensitivity to food. Conversely, temporal activation of MIP neuron markedly reduced the food intake and BW, and blunted the sensitivity of the starved flies to food as if they have been satiated. Shortly after termination of MIP neuron activation, the reduced BW reverted to the normal level along with a strong feeding rebound. Together our results reveal the switch-like role of the MIP pathway in feeding regulation by controlling satiety. [BMB Reports 2016; 49(3): 137-138]

Appropriate regulation of feeding behaviors is critical for the survival of animals. Disruption of feeding regulation results in feeding-related disorders, including obesity and hyper- or hypophagia. The feeding behaviors are primarily shaped by two motivational states - hunger and satiation. Hunger cues an animal to seek food sources and ingest nutritive food, while satiation signals a feeding animal to cease further ingestion of food to prevent animals from excessive energy gain.

Recent studies using Drosophila have shed much light on animal feeding behaviors, specifically on those driven by hunger. Several lines of evidence have suggested that hungry fruit flies are capable of evaluating caloric values of sugars and selecting nutritive sugars over zero-calorie ones. Dh44 (the homolog of cortisol releasing hormone; CRH)-expressing neurons are activated by nutritive sugars but not by zero-calorie sugars, to promote feeding. Another group of neurons expressing the Gustatory receptor 43a (Gr43a) in the brain detects hemolymph fructose levels after a meal, to promote feeding in hungry flies. More recently, a subset of serotonergic neurons was shown to evoke several traits of hunger responses when activated, indicating that these neurons mediate the representation of hunger in Drosophila.

In mammals, feeding behaviors are regulated by anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AGRP) neurons in the hypothalamic arcuate nucleus. Genetic ablation of POMC markedly elevates both food intake and BW. Conversely, activation of AGRP neurons that also express the neuropeptide Y (NPY), promotes feeding activity. As in mammals, the neuropeptide F, an orthologue of mammalian NPY, has been shown to promote feeding activity and hunger-driven behaviors in Drosophila. Unlike POMC system in mammals, some known anorexigenic neural pathways in Drosophila appear dispensable for BW homeostasis.

To identify an anorexigenic neural pathway that functions critically for the BW regulation in Drosophila, we performed a genetic screen using the GAL4-UAS system. A collection of neuropeptide-GAL4 driver lines that label neuropeptide neurons had a neural silencer line bearing UAS-tetanus toxin (UAS-TNT). In the screen, the progenies from the cross would harbor individual neuropeptide neurons selectively silenced, depending on the driver. Our working hypothesis was that flies with silenced neuropeptide neurons critical for satiety control would show notable BW increase. Indeed, we were able to select a GAL4 driver line that elicited a conspicuous BW increase in both sexes when crossed to UAS-TNT. This GAL4 driver was composed of GAL4 fused with the 5’ upstream regulatory sequence of the gene encoding MIP.

Flies with silenced MIP neurons (MIP＞TNT) showed an ab-normal elevation of food intake, accompanied by a marked BW increase. The increased BW of MIP＞TNT flies was completely restored to normal levels in a restricted feeding condition, indicating the strong correlation between feeding and BW regulation. Having shown that silencing MIP neurons increased BW through elevated food intake, we sought to artificially activate MIP neurons using a thermogenetic approach to examine whether BW and food intake conversely decreased upon MIP neuron activation. To do so, we employed TRPA1 that excites neurons in response to warm temperature at 30℃ when it is ectopically expressed. Flies harboring UAS-TRPA1 and MIP-GAL4 (MIP＞TRPA1) incubated at 30℃ showed a dramatic decrease in BW, and this decrease completely reversed back to normal upon termination of neural activation, by incubating the MIP＞TRPA1 flies at 18℃. Similarly, MIP＞TRPA1 flies exhibited strongly suppressed food intake at 30℃; however, the suppressed food intake became restored to normal level with a strong feeding rebound upon neural termination, indicating the switch-like role of MIP neurons in regulation of food intake. Together, these results revealed a negative correlation between MIP neuron activity and food intake in BW control.

Next, we and our colleagues sought to visualize the MIP neurons using the MIP-GAL4 transgene, and a highly specific anti-MIP antibody and MIP mRNA anti-sense probe. Using double-labeling experiments, we found 52 authentic MIP neurons positive for both anti-MIP antibody and MIP mRNA anti-sense probe in the central nerve system. Notable innervations by the MIP neurons were observed in the primary brain structures, including the antennal lobe and subesophageal zone, important for olfactory and gustatory perception of food. Remarkably, the expression of Mip RNAi driven by MIP-GAL4 eliminated most of the anti-MIP antibody signals and elicited a BW increase, indicating that specific subsets of MIP-GAL4 neurons expressing MIP are critical for BW control. To delicately define the neurons responsible for BW control, we employed subset-specific GAL80 (the suppressor of GAL4) lines and identified that Cha-GAL80 fully suppressed the BW phenotype of MIP＞TNT flies. This result indicates that the Cha-GAL80 tags the subset neurons, which are important for BW control. By comparing the expression patterns of Cha-GAL80 with other GAL80 lines, we were further able to identify a cluster of neurons in the brain presumed to be critical for Drosophila BW control.

The observation that the activity of MIP neurons controlled BW and food intake, and that MIP was expressed throughout the MIP neurons, led us to expect the significant role of MIP in BW and food intake. However, MIP has previously been reported as a potent ligand for sex peptide receptor (SPR), and thus its role in sexual behavior was anticipated. Furthermore, a recent study showed that the MIP-SPR pathway is also involved in the maintenance of sleep behavior in Drosophila. In our study, we and our colleagues for the first time generated a null mutation for MIP and examined its role in food intake and BW. Similar to MIP＞TNT flies, the mutants lacking MIP expression showed a significant increase in BW and food intake. These defects were rescued by genetic restoration of MIP expression in the mutants, but not by genetic manipulation of SPR gene expression. These results suggest that MIP in MIP neurons plays a critical role in BW regulation independently of SPR.

Finally, along with our colleagues, we attempted to address the physiological meaning of the MIP pathway-mediated BW regulation in animals, by testing the possibility that the MIP pathway controls satiety. Satiety is characterized by the animals’ blunted peripheral sensitivity to food. For example, the satiated flies exhibit decreased olfactory attraction to food odors and/or reduced proboscis extension reflex (PER) to sugars as satiety responses. Using these behavioral paradigms, we quantified satiety responses of flies and examined the role of the MIP pathway in satiety control. Flies with silenced MIP neurons and/or MIP mutation showed significantly enhanced olfactory attraction to food odors. Consistent with this result, electrophysiological recordings on olfactory sensilla of the MIP＞TNT flies showed elevated neural responses to food odors. Likewise, suppression of the MIP pathway in the satiated flies greatly elevated the PER responses to sucrose, as if the flies had been starved. Conversely, activation of the MIP neurons in the starved flies completely blunted the olfactory and gustatory responses, as if the flies had been satiated. These results strongly support that the MIP pathway is required, and is sufficient for inducing satiety responses.

Based on these data, we propose a model in which the MIP neurons in the brain induce satiety and suppress food intake to maintain a constant BW in a MIP neuropeptide-dependent mechanism.