Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila

Abstract

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. Here, we report on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. We found that overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppressed the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons led to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, we provide evidence that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. Our study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways.

Fig. 1.

The dS6K activity in DILP neurons mediates hunger regulation of both food preference and ingestion rate. Transgenic analysis of dS6K activity was performed by using the Drosophila GAL4/UAS binary expression system (40). UAS-dS6K^DN and UAS-dS6K^ACT encode a dominant-negative and constitutively active form of dS6K, respectively. All of the fly lines used are in a w background. All of the controls in this and other figures are isogenic to the relevant experimental larvae except for the transgene tested. (A) Quantification of liquid and solid food intake by synchronized w¹¹¹⁸ larvae (74 h after egg laying, AEL) in response to increasing food deprivation. The number of larval mouth-hook contractions within a 30-s interval was measured. We found that larvae display virtually identical feeding responses to various liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions (see Fig. 6). (B) DILP neurons expressing GFP in dilp2-gal4 × UAS-GFP larvae. The dilp2-gal4 driver selectively directs a GFP reporter expression in the two clusters of medial neurosecretory cells, as indicated by arrowheads. No GFP expression was detected in cells of other tissues including the gut, imaginal discs, or salivary glands. (Magnification: ×200.) (C and D) Synchronized third-instar larvae were withheld from food for 0 or 120 min before the assay. Control larvae: w × UAS-dS6K^DN; w × UAS-dS6K^ACT. Experimental larvae: dilp2-gal4 × UAS-dS6K^DN; dilp2-gal4 × UAS-dS6K^ACT. At least 30 larvae per group were assayed in three separate trials. Fed larvae expressing dS6K^DN in DILP neurons showed significant increases in the feeding of liquid and solid food relative to controls (P < 0.0001), whereas those overexpressing dS6K^ACT showed no changes in the feeding responses (P > 0.34). Fasted larvae expressing dS6K^ACT in DILP neurons showed attenuated feeding of the liquid and solid food relative to controls (P < 0.0001), but those overexpressing dS6K^DN showed no altered feeding responses (P > 0.16). At least 20 larvae per group were assayed in three separate trials. All statistical analyses were performed by using ANOVA. Error bars are the SEM.

Fig. 2.

Overexpression of DILPs suppresses hunger-driven feeding activities. The elav-gal4 flies are in a w background; the UAS-dilp2, dilp3, and dilp4 lines are in a y w background. Overexpression of DILPs in the larval nervous system was directed by elav-gal4. Larvae were fasted for 120 min before the assays. The controls (elav-gal4 × UAS-GFP,w × UAS-dilp2, dilp3, and dilp4) displayed normal increases in feeding rate and motivated intake of the less preferred solid food. In contrast, the experimental larvae overexpressing dilp2 or dilp4 but not dilp3 showed significantly attenuated hunger responses (P < 0.0001 and P > 0.05, respectively). It still remains unclear why DILP3, which was shown to increase body size (23), is ineffective in attenuating feeding response. It is possible that DILP3 may be a weaker ligand to neural dInR. At least 20 larvae per group were assayed in three separate trials.

Fig. 3.

The dInR and dS6K activities in NPFR1 neurons selectively regulate food selection. The npfr1-gal4 is in a y w background, whereas all of the UAS line are in a w background. (A and B) UAS-dInR^DN and UAS-dInR^ACT each encodes a dominant-negative and a constitutively active form of dInR. UAS-dp110 and UAS-dPI3K^DN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively. UAS-dPTEN encodes WT dPTEN. Control larvae: yw × UAS-dInR^DN, UAS-dInR^ACT, UAS-dp110, UAS-dPI3K^DN, and UAS-dPTEN. Experimental larvae: npfr1-gal4 × UAS-dInR^DN, UAS-dInR^ACT, UAS-dp110, UAS-dPI3K^DN, and UAS-dPTEN. At least 20 larvae per group were assayed in three separate trials. Fed larvae expressing transgenes (dInR^DN, dPI3K^DN, or dPTEN) that suppress dInR signaling in NPFR1 neurons showed significant feeding of the solid food (P < 0.0001), whereas deprived larvae expressing transgenes (dInR^ACT or Dp110) that enhance dInR signaling displayed attenuated feeding of the solid but not liquid food (P < 0.0001). (C and D) Control larvae: yw × UAS-dS6K^DN, yw × UAS-dS6K^ACT. Experimental larvae: npfr1-gal4 × UAS-dS6K^DN, npfr1-gal4 × UAS-dS6K^ACT. Larvae expressing dS6K^DN in NPFR1 cells showed significant feeding of the solid but not liquid food (P < 0.0001) without deprivation, whereas fasted larvae expressing dS6K^ACT displayed attenuated feeding of the solid but not liquid food (P < 0.0001).

Fig. 4.

The NPF/NPFR1 pathway acutely mediates hunger regulation of food preference. The UAS-npfr1 and UAS-ANF-GFP are in the y w background, whereas the elav-gal4, appl-gal4, MHC82-gal4, UAS-shi^ts1, and UAS-npfr1^dsRNA are in a w background. (A–C) NPFR1-overexpressing larvae fasted for 0, 30, or 120 min were assayed. The rate of mouth-hook contractions was scored individually (n = 15–20 per group for each of three trials). Among the fed larvae, the experimental group (npfr1-gal4 × UAS-npfr1) showed significantly higher activity of extracting agar-embedded glucose relative to controls (npfr1-gal4 × y w, npfr1-gal4 × UAS-ANF-GFP, and y w × UAS-npfr1; P < 0.0001), whereas the ingestion rate of the liquid food remained unchanged (P > 0.25). The enhancement of motivated intake of the solid food was also observed in NPFR1-overexpressing larvae fasted for 30 min (P < 0.001), but the difference became insignificant after 120 min (P > 0.86). (D) Larvae expressing npfr1 dsRNA in NPFR1 cells and the nervous system were tested for their feeding responses to liquid and solid food. Larvae were fasted for 120 min before the assay. The control larvae (w¹¹¹⁸, npfr1-gal4 × w, y w × npfr1^dsRNA, w × UAS-npfr1^dsRNA, elav-gal4 × w, and MHC-82-gal4 × UAS-npfr1^dsRNA) showed enhanced feeding responses, whereas the experimental larvae (npfr1-gal4, elav-gal4, and appl-gal4 × UAS-npfr1^dsRNA) were deficient in feeding response to the solid but not liquid food (P < 0.0001; n > 20 per group). The UAS-npfr1^dsRNA line was previously shown to reduce npfr1 transcript levels and NPFR1 signaling activity (32). (E and F) UAS-shi^ts1 encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). Larvae were fasted for 120 min before the assay (n > 20 per group for each of three trials). At 23°C, both experimental larvae (npf-gal4 and npfr1-gal4 × UAS-shi^ts1) and control larvae (y w × UAS-shi^ts1, npf-gal4 × w, and npfr1-gal4 × w) displayed normal food response (P > 0.45). At 30°C, the experimental larvae showed deficits in motivated feeding response to the solid food but not liquid food (P < 0.0001), whereas the controls remained normal. The feeding behaviors of control larvae at both temperatures were also similar (P > 0.08).

Fig. 5.

A model for the hunger regulation of adaptive feeding behaviors in Drosophila larvae. Our results suggest that DILP2 neurons, and possibly together with DILP4 neurons, negatively regulate two downstream hunger-responsive feeding systems: an NPF/NPFR1-dependent pathway specialized for motivated feeding and an NPF/NPFR1-independent pathway for general enhancement of feeding rate. In DILP neurons, dS6K activity is likely to positively regulate DILP synthesis and/or release. In fed larvae, a relatively high level of DILP signaling suppresses the two downstream pathways. In fasted animals, hunger stimuli down-regulate dS6K activity in DILP neurons, which in turn leads to decreased DILP signaling and therefore disinhibition of the two pathways. The former overrides the high threshold set by the default pathway, enabling hungry animals to engage in motivated foraging and food selection. The latter enhances feeding rate, allowing animals to compete effectively for limited food sources. DILPs negatively regulate the NPFR1 pathway through the dInR/dPI3K/dS6K pathway in NPFR1 neurons. Our data also implicate the presence of a separate default pathway for ad libitum feeding of higher-quality foods (baseline feeding) by fed larvae. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in fed larvae did not affect baseline feeding in the presence of the liquid food (see Figs. 1, 2, 3 and data not shown).