A Leptin Analog Locally Produced in the Brain Acts via a Conserved Neural Circuit to Modulate Obesity-Linked Behaviors in Drosophila  Jennifer Beshel, Josh Dubnau, Yi Zhong  Cell Metabolism  Volume 25, Issue 1, Pages 208-217 (January 2017) DOI: 10.1016/j.cmet.2016.12.013 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 25, 208-217DOI: (10.1016/j.cmet.2016.12.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Brain-Based Upd1 Regulates Responsiveness to Food Cues, Food Intake, Weight, and Sensitivity to an Obesogenic Environment (A) Antibody staining shows upd1-immunopositive cells in neural tissue (red, background subtracted). nc82 staining is in blue. Scale bar, 20 μm. (B) Upd1 antibody fluorescence at the level of the cell body is visibly reduced in fed flies, indicating increased upd1 signaling in the fed state. (C) Quantified fluorescence intensities for upd1+ cells in fed and starved flies show significantly lower accumulation of somatic upd1 in fed flies. (D) Fed adult flies with brain-specific knockdown of upd1 using the elav-gal4 and two independent upd1-RNAi lines show starved-like attraction to a yeast odorant food cue. Example 2D histograms show cumulative fly counts across the four-field olfactometer with one quadrant odorized with the attractive food cue yeast. Higher-occupancy levels in the odorized quadrant reflect greater attraction to the food cue. Yeast-odorized quadrant was randomized. Images reflect the location distributions for the entire 10 min testing period and are altered to depict the odorized quadrant as the right quadrant. (E) Fed flies lacking upd1 in neural tissue are as attracted to the odorant food cue as starved flies. (F) CAFE assay demonstrates increased food intake. (G and H) Upd1-deficient flies (G) are heavier on standard lab media and (H) show increased fat deposition. (I) Pan-neuronal upd1 knockdown leads to disproportional increases in weight when exposed to a high-fat diet. Example images of fly size after exposure to a high-fat diet. (J) Weights after high-fat diet. (K) Percentage weight gain after high-fat feeding relative to weight on standard media (see G). (L–N) Normal-fed fly behavior is rescued with upd1, human leptin, and upd2 in olfactometer (L), CAFE (M), and weight (N) assays. ∗p < 0.05, ∗∗p < 0.01; different from parental controls, except for B2, where significance values refer to fed versus starved; ns, not significant. n = 8–12 groups of flies for each condition. Each value is mean ± SEM. See also Figures S1 and S2. Cell Metabolism 2017 25, 208-217DOI: (10.1016/j.cmet.2016.12.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Obesity Phenotypes Can Be Both Rescued and Elicited Acutely (A) Schematic of temperature shift experimental design. Genotypes and ultimate effect on upd1. (B) Flies carrying temperature-sensitive Gal80ts and elav-Gal4 driving UAS-upd1 RNAi raised in 30°C lack brain-based upd1 throughout development. Shift to 18°C after eclosion reinstates normal upd1 function in the adult. (C) The same genotype raised in 18°C and shifted to 30°C induces upd1 knockdown post-developmentally. (D) Obesity phenotypes are rescued in the adult. Example 2D histograms show cumulative fly counts across the four-field olfactometer. Odorized quadrant was randomized. Images reflect the location distributions for the entire 10 min testing period and are altered to depict the odorized quadrant as the right quadrant. (E) Fed flies with upd1function restored in neural tissue as adults behave in a satiety-state-appropriate manner. (F) CAFE assay demonstrates normal food intake. (G) Upd1-restored flies are of normal weight. (H) Obesity phenotypes are induced post-developmentally. Example 2D histograms of behavior in the olfactometer assay (as in D). (I) Fed flies with upd1 knockdown induced in adulthood are as attracted to the odorant food cue as flies lacking upd1 throughout development. (J) CAFE assay demonstrates commensurate food intake. (K) Acute upd1 deficiency leads to heavier flies. ∗p < 0.05, ∗∗p < 0.01; different from parental controls. n = 8–12 groups of flies for each condition. Each value is mean ± SEM. Cell Metabolism 2017 25, 208-217DOI: (10.1016/j.cmet.2016.12.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Manipulation of Domeless Receptors on Npf Cells Recapitulates Obesity-like Phenotypes Observed in Flies Lacking Upd1 in Neural Tissue (A) Antibody staining shows npf-immunopositive cells (red) overlap with the domeless expression pattern. GFP staining is in green. Scale bar, 20 μm. (B) The same npf+ cells co-localize with the npf-GAL4. (C) Fed adult flies with npf-specific knockdown of domeless using the npf-gal4 and two independent dome-RNAi lines show starved-like attraction to a yeast odorant food cue. Example 2D histograms show cumulative fly counts across the four-field olfactometer as in Figure 1D. (D) Fed flies lacking domeless in npf cells behave as though starved to the odorant food cue. (E) CAFE assay demonstrates increased food intake. (F and G) Flies (F) are heavier on standard lab media and (G) show increased fat deposition when domeless knockdown is targeted specifically to npf cells. (H) Npf-specific domeless knockdown leads to increased sensitivity to a high-fat diet. Example images of fly size after exposure to a high-fat diet. (I) Weights after high-fat diet. (J) Percentage weight gain after high-fat feeding relative to weight on standard media. ∗p < 0.05, ∗∗p < 0.01; different from parental controls, except for B2, where significance values refer to fed versus starved; n = 8–12 groups of flies for each condition. Each value is mean ± SEM. See also Figure S3. Cell Metabolism 2017 25, 208-217DOI: (10.1016/j.cmet.2016.12.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Brain-Based Upd1 Acts via Domeless Receptors on Npf Cells to Modulate Satiety-State Dependence of Food-Odor-Evoked Npf Activity (A) In vivo two-photon imaging of npf calcium activity in response to food odor stimulation. Grayscale images show single dorsomedial npf cell (dashed outline) expressing gCaMP3 under npf-gal4 control; pseudocolored images show yeast odor-evoked activity in fed (left) and starved (right) flies. Scale bar, 1 μm. (B) Mean ΔF/F time course of the npf neuron for fed (red) and starved (black) flies in response to yeast (blue) and apple cider vinegar (orange). Horizontal bars represent the 3 s odor delivery period. (C) Peak ΔF/F odor response (0–5 s after stimulus onset). The npf neuron responds to food odors and this response is elevated after starvation. (D) Flies with upd1 knockdown targeted to neural tissue lose the satiety-state-dependent modulation of odor-evoked responses. Example images from flies also carrying elav-gal4 and UAS-upd1 RNAi transgenes. (E) Mean ΔF/F time course of the npf neuron. (F) Peak ΔF/F odor response. (G) FB-targeted upd2 knockdown does not affect satiety-state-dependent modulation of odor-evoked response. Example images from flies also carrying cg-gal4 and UAS-upd2 RNAi transgenes. (H) Mean ΔF/F time course of the npf neuron. (I) Peak ΔF/F odor response. (J) Npf-targeted domeless receptor knockdown eliminates satiety-state dependence. Example images from flies also carrying UAS-domeless RNAi transgenes. (K) Mean ΔF/F time course of the npf neuron. (L) Peak ΔF/F odor response. ∗p < 0.05, ∗∗p < 0.01 fed versus starved; ns, not significant. n = 10 cells from 10 flies for each satiety state. Each value is mean ± SEM. See also Figure S4. Cell Metabolism 2017 25, 208-217DOI: (10.1016/j.cmet.2016.12.013) Copyright © 2017 Elsevier Inc. Terms and Conditions