Cell Metabolism, Volume 24

Supplemental Information

Sucralose Promotes Food Intake

through NPY and a Neuronal Fasting Response

Qiao-Ping Wang, Yong Qi Lin, Lei Zhang, Yana A. Wilson, Lisa J. Oyston, JamesCotterell, Yue Qi, Thang M. Khuong, Noman Bakhshi, Yoann Planchenault, Duncan T.Browman, Man Tat Lau, Tiffany A. Cole, Adam C.N. Wong, Stephen J.Simpson, Adam R. Cole, Josef M. Penninger, Herbert Herzog, and G. Gregory Neely

Supplemental Figures and Legends

Figure S1. A Synthetically Sweetened Diet Has No Major Effect on Metabolic Parameters and Acts Independent of

the Gut Microbiome, related to Figure 1. (A-B) Sucralose effect on appetite was not altered by (A) tetracycline (50

µg/ml) or (B) in germ-free flies; n ≥ 12 replicates (5 animals per replicate for all feeding experiments). (C-F) The

sweetened diet had no significant effect on (C) body weight (n = 4), (D) triglycerides (n ≥ 8), (E) glycogen (n ≥ 20), (F)

or hemolymph glucose levels (n ≥ 8), (10-20 animals per replicate). (G) Pretreated with L-glucose did not cause increased

activity (n=32 flies). All data represented as mean ± S.E.M., unpaired t-test *, p < 0.05, and **, p < 0.01. n.s., not

significant.

Figure S2. A Synthetically Sweetened Diet Promotes Hunger via Octopamine and Dopamine Pathways, related to

Figure 4. (A) RNAi Knockdown of InR in octopaminergic neurons blocked the sucralose effect on PER, n ≥ 3 replicates

(10-13 animals per replicate for all PER experiments). (B) Prolonged TrpA1 activation in octopaminergic neurons was

not sufficient to mimic the sucralose effect on food intake, n ≥ 7. (C) Octopamine receptors Octβ1R, Octβ2R, and Octβ3R

were not required for increased feeding after the sucralose-sweetened diet, n ≥ 14. (D) Oamb mutants failed to increase

feeding in response to a sweetened diet, n ≥ 17. (E) Prolonged TrpA1 activation in dopaminergic neurons was not

sufficient to mimic the sucralose effect on food intake, n ≥ 7. (F) Knockdown of InR in dopaminergic neurons blocked the

sucralose effect on PER, n ≥ 3. (G) Knockdown of Oamb in dopaminergic neurons blocked the sucralose effect on PER, n

≥ 3. (H) Dopamine receptors DopR1 and Dop2R were not required for increased feeding after a sucralose-sweetened diet,

n ≥ 21. (I) Increased feeding in response to a sweetened diet was impaired DopR2 and DopEcR mutants, n ≥ 20. (J)

Knockdown of DopR2 in octopaminergic and insulin-producing neurons did not block sucralose effect on PER, n ≥ 21.

All data represented as mean ± S.E.M. One-way ANOVA with Turkey’s multiple comparisons test was used for feeding

and two-way ANOVA with Sidak's multiple comparisons test was used for all PER experiments, *, p < 0.05, **, p <

0.01, ***, p < 0.001, and ****, p< 0.0001. n.s., not significant. Also see Table S1.

Figure S3. Synthetically Sweetened Food Works Through the NPF System to Alter Sweet Taste Perception,

related to Figure 5 (A) RNAi knockdown of DopR2 in Gr64f+ neurons did not block the sucralose effect on food intake,

n ≥ 21. (B) Control flies UAS-DopR2 RNAi/+ did not show impaired sucralose effect by PER, n ≥ 3. (C) RNAi

knockdown of DopR2 in NPF+ neurons increased basal PER response, n ≥ 3. (D) Synaptic outputs from NPFR+ was

required for increased food intake after sucralose diet, n ≥ 14. (E) Pan-neuronal knockdown of NPFR blocked the

sucralose effect on PER, n ≥ 3. (F) Knockdown of NPFR in Dilp2+ neurons did not block the sucralose effect on food

intake; n ≥ 14 (G) Knockdown of InR in NPF+ neurons blocked increased PER, n ≥ 3. All data represented as mean ±

S.E.M. One-way ANOVA with Turkey’s multiple comparisons test was used for feeding and two-way ANOVA with

Sidak's multiple comparisons test was used for all PER experiments, unpaired t test was used for S50 comparisons **, p

< 0.01, ***, p < 0.001, and ****, p< 0.0001. n.s., not significant. Also see Table S1.

Figure S4. Neuronal AMPK is Activated by a Sweetened Diet and is Required for the Effects on Food Intake and

PER Response, related to Figure 6. (A) Pan-neuronal expression of kinase dead AMPK prevented the sucralose effect

on feeding, n ≥ 14. (B) AMPK knockdown or (C) expression of kinase dead AMPK in insulin-producing neuron,

octopaminergic and Gr64f+ neurons did not suppress the sucralose effect on feeding. (D) Expression of kinase dead

AMPK in dopaminergic and NPF neurons prevented sucralose effect on feeding, n ≥ 14. (E) Control UAS-AMPK RNAi/+

flies show intact sucralose response by PER, n ≥ 3. All data represented as mean ± S.E.M., One-way ANOVA with

Turkey’s multiple comparisons test was used for feeding data and two-way ANOVA with Sidak's multiple comparisons

test was used for all PER experiments, *, p < 0.05, **, p < 0.01, and ****, p<0.0001. n.s., not significant. Also see

Table S1.

Figure S5. Sucralose Promotes Hunger Through a Fasting Response Pathway, related to Figure 7. (A-C) Blockage

of synaptic output from (A) octopaminergic-, (B) dopaminergic- and (C) NPF- neurons impaired enhanced PER after

fasting, n ≥ 3. (D) Control flies nSyb-Gal4+, Tdc2-Gal4+, TH-Gal4/+, NPF-Gal4/+, Gr64f-Gal4+, UAS-InR RNAi/+,

UAS-Oamb RNAi/+, UAS-DopR2 RNAi/+, UAS-NPFR RNAi/+, and UAS-AMPK RNAi/+ show intact PER sensitization

after fasting, n ≥ 3 (E) Pan-neuronal knockdown of AMPK prevented enhanced PER caused by fasting, n ≥ 3 (F) Fasting

did not cause significant spike frequency increase in response to 100mM [sucrose] stimulation in WT flies, n = 9-14

animals. All data represented as mean ± S.E.M., unpaired t-test was used for electrophysiology data and two-way

ANOVA with Sidak's multiple comparisons test was used for all PER experiments, *, p < 0.05, **, p < 0.01, ***, p <

0.001, and ****, p< 0.0001. n.s., not significant. Also see the Table S1C.

Supplemental Table Legends

Table S1. The basal food intake, and S50 of sucralose PER and starvation PER of the test genotypes, Related to

Figure 3-7. (A) Basal food intake for genes involved in sucralose response. Food intake was measured during 24h and

expressed as nL/fly/day. One-way ANOVA with Turkey's multiple comparisons test was used for statistical analysis. p <

0.05 was considered significant (B) The S50, the sucrose concentration that cause 50% of PER response, of the test

genotypes with or without sucralose treatment. (C) The S50 of the test genotypes with or without fasting. Genotypes,

replicates (N), Mean, STDEV and SEM are provided (Excel file).

Table S2. Sucralose-regulated transcripts detected by RNA Sequencing, Related to Figure 3. RPKM values for each

transcript identified from RNA sequencing fly head before (Control) or after 6 days sucralose-sweetened (Sucralose)

diets. Flybase ID, Transcript size, expression, RPKM, log2 ratio (Sucralose RPKM/Control RPKM), direction of

regulation, p-value and false discovery rate (FDR) of each transcript are included. The red color indicates up-regulated

genes and blue color down regulated genes (Excel file).

Supplemental Experimental Procedures

Fly Strains

Fly stocks were maintained on standard diet and were raised in 25°C incubator with a 12/12 light/dark cycle. Tdc2-Gal4

(Cole et al., 2005)(#9313), TH-Gal4 (Friggi-Grelin et al., 2003) (#8848), Dilp2-Gal4 (Rulifson et al., 2002)(#37516),

UAS-TeTxLC.TNT (UAS-TNT, #28838) and UAS-TeTxLC.IMP TNT (UAS-iTNT, (#28841) (Sweeney et al., 1995), UAS-

Octβ1R-IR (#50701), UAS-Octβ2R-IR (#34673), UAS-Octβ3R-IR (#31108), UAS-Oamb-IR (#31171, #31233), UAS-InR-

IR (#31594, #35251), DopR2MB05108 (#24743), UAS-AMPKdead (#32112), and UAS-AMPK-IR (#25931, #32371) were

obtained from the Bloomington Stock Center. NPF-Gal4 and NPFR-GAL4 (Wu et al., 2003) were from Ping Shen. UAS-

Dicer 2, nSyb-Gal4 (III) (Dietzl et al., 2007)was from Partrik Verstreken. Gr64f-Gal4 (Weiss et al., 2011) was from Alex

Keene. Tub-Gal80ts was from Bruno Van Swinderen. Wild type w1118 was from Hugo Bellen. ΔGr5a and ΔGr64a

(Dahanukar et al., 2007) was from Greg Suh. UAS-NPFR-IR (Wen et al., 2005), Oamb584 (Lee et al., 2003) and

NPFRc01896 (Krashes et al., 2009) were from Scott Waddell. UAS-TrpA1 (Inagaki et al., 2012) and DopEcRc02142 (Ishimoto

et al., 2013) were from David Anderson. UAS-NPFR-IR (KK107663) UAS-DopR1-IR (KK107058), UAS-DopR2-IR

(KK105324, GD3391), UAS-DopD2R-IR (GD11470), and UAS-DopEcR-IR (KK103444) were from the VDRC.

Proboscis Extension Response Assay

For PER responses after sucralose sweetened diet, flies were pretreated with control or sucralose-sweetened diet for 6

days, and then PER assay was performed as described (Masek and Keene, 2013). For fasting PER, 3 to 7 day old flies

were fed or wet-starved for 6 hours and then PER assay was performed. In all PER assays, flies were first tested with

water before providing sucrose solutions. Flies that responded to water were discarded. Taste bristles on the front legs

were stimulated by sucrose solution twice and PER responses were scored as follows: no extension with two tastants

exposures= 0 and full extension with at least one tastants exposure = 1. At least 3 groups were tested for each genotype.

S50 Estimation

The S50, the sucrose concentration that induces 50% PER, was estimated using logistic sigmoid function:

f (x)=L/1+e-k(Log2

x-Log2

S50

) based on a previous method(Inagaki et al., 2014). Here are the definitions:

1. L, theoretical maximum PER value, here 100% is used

2. k, slope of the curve

3. x, sucrose concentration

All S50 estimations were performed in R package using “nls” function. The R scripts will be provided by request.

Metabolic Parameters

Triglyceride assay: was adapted from (Pospisilik et al., 2010). 10 male flies were weighted and homogenised in 200µl

dH2O on ice, then sonicated for 10s using a probe sonicator on ice. After sonication, 800µl ice-cold dH2O was added and

mixed thoroughly. 50µl of the mixture was used to determine the triglycerides using the Roche triglycerides kit

(11730711216) under the manufacture’s instructions. Triglycerides were normalized to body weight. Glucose assay: was

adapted from (Miyamoto et al., 2012). 10 male flies heads were homogenized in 200µl dH2O on ice and then 800µl ice-

cold H2O was added then mixed thoroughly. The mixture was centrifuged at 13000 rpm for 2 min at 4°C and 50 µl of

mixture was used to determine the [glucose] using Thermo Infinite Glucose kit (TR15421) as the manufacture’s

instructions and 25 µl of mixture was used to determine protein using BCA protein assay kit (Thermo Fisher Scientific).

Glycogen assay: [glycogen] was determined by measuring the glucose degraded from glycogen using amyoglucosidases.

10-15 male flies were homogenized and dissolved in 1M KOH solutions. After twice 95% ethanol extraction, the pellet

was resuspended 1ml amyoglucosidase reaction buffer (0.3 mg/ml in 0.25 M acetate buffer, pH4.75) and incubated in the

37°C shaking incubator overnight and a glucose assay was performed. Oral Glucose tolerance test (Haselton et al., 2010):

Flies were wet-starved for 16 hours and then fed with 10% glucose for 1 hour. Flies were then wet-starved for 30 or 60

min, collected, and a glucose assay was performed.

Locomotor Activity and Sleep Assay

Locomotor activity and sleep were determined using the DAM (Drosophila Activity Monitor) system (Trikinetics), which

records activity when a fly crosses an infrared beam. 3 to 7 days old flies were loaded into tubes that had control diet or

sucralose diet housed in 12 hour light/dark (LD) cycles at 25°C and monitored for 7 days. Locomotor activity was

calculated from extracting data at 30 min bins. Sleep analyses were performed by using data extracted at 5 min bins

similar to previous studies (Pfeiffenberger et al., 2010).

Electrophysical Recording

Tip recordings were performed as previously described (Dahanukar et al., 2001; Hiroi et al., 2002; Hodgson et al., 1955).

All recordings were performed on 10 to 13 day-old flies that were pretreated with or without sucralose for 6 days, or 4 to

7 day-old flies with or without 6h wet starvation. 3-5 L-type labellar bristles were recorded on each fly. The recording

electrode (tip diameter, 10–12 µm) was filled with 20 or 100mM [sucrose] solution. Each chosen L-type bristle was first

tested by 1mM KCl to confirm its healthiness, followed by application of sucrose in 30mM tricholine citrate (TCC,

Sigma-Aldrich as electrolyte). Labellar taste sensilla were stimulated up to 4 seconds, To avoid adaptation, sensilla were

stimulated for 4 seconds each time and allowed to recover for >2 minutes before applying another stimulus.

Signals were acquired using an AxonClamp 900A amplifier and digitized with a 1400A D-A converter (Molecular

Devices) at sampling rate of 10 kHz, filtered at 3 kHz. Electric signals were further amplified and filtered by a second

amplifier (CyberAmp 320, Axon Instrument, Inc., USA, gain X 100, eighth order Bessel pass-band filter 1600 Hz). Data

was analyzed using the Clampfit 10 software (Molecular Devices). Spikes between 0 and 2 s after initiation of stimuli

were counted as firing frequency evoked by the concentration of sucrose. The mean value of spikes was calculated on 3-5

bristles recorded on each fly as one statistical sample. The mean ± SEM in figures and text were based on number of flies.

RNA sequencing

Fifty fly heads were collected at day 0 or day 6 (6 days sucralose treatment). RNA was extracted using Trizol (Life

Technologies, #10296010) and then sequenced by BGI. Total RNA samples were first treated with DNase I to degrade

any possible DNA contamination. Then mRNAs were enriched using oligo(dT) magnetic beads and short fragments

(about 200 bp) were generated. The first strand of cDNA was synthesized by using random hexamer-primer and then

buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA

was purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition was then performed.

Finally, sequencing adaptors were ligated to the fragments and enriched by PCR amplification. Agilent 2100 Bioanaylzer

and ABI StepOnePlus Real-Time PCR System were used to qualify and quantify of the sample library. The library

products were sequenced via Illumina HiSeqTM 2000.

Bioinformatics Analysis.

Gene expression level was calculated using RPKM (Reads Per kb per Million reads) (Mortazavi et al., 2008). The

formula use was: RPKM=106C / (NL/103), C is number of reads that uniquely aligned to a gene, N is total number of

reads that uniquely aligned to all genes, and L is number of bases of a gene. Transcripts with Log2 ratio>1, FDR<0.001

were considered differentially regulated.

Western Blot

Western blot was performed according to a standard protocol. Fly heads were collected and homogenized in PBS with

Roche protease inhibitors cocktail. Protein was denatured and run in 10% SDS-PAGE. Protein was transferred to

membrane using semi-dry machine (Bio-Rad). The membrane was blocked by 5% milk in TBS. The membrane was

incubated overnight with primary antibodies diluted in 1:1000 in 5% BSA in TBST (Tween, 0.1%). Rabbit-anti-AMPKα

(Cell signaling, #2532), Rabbit-anti-pAMPKα172 (Cell signaling, #2535). Secondary antibodies were used at a dilution

of 1 in 10000 (BioRad). Qualification was performed using Image J software.

qPCR

1µg of mRNA was reversely transcribed into cDNA using SuperScript® III First-Strand Synthesis System (Invitrogen).

All primers used for qPCR that have been prescreened for efficiency and specificity. RT-PCR was performed using

SensimixTM probe kit (Bioline). The program is following: 95°C 10 min, 40 cycles of 95°C, 15s; 55°C, 15s; 72°C, 15s.

The reactions were run on LightCycle® 480 (Roche). The gene expression was normalized to the reference gene rp49.

RNAi Knockdown efficiency ranged from ~30% (InR and AMPK) to 70% (Oamb, DopEcR).

Mouse Food Intake

All research and animal care procedures were approved by the Garvan Institute / St. Vincent’s Hospital Animal Ethics

Committee and were in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific

Purpose. Mice were housed under conditions of controlled temperature (22°C) and illumination (12-h light cycle, lights

on at 07:00 h). All mice were fed a normal chow diet (8% calories from fat, 21% calories from protein, 71% calories from

carbohydrate, 2.6 kcal/g; Gordon’s Speciality Stock Feeds, Yanderra, NSW, Australia) ad libitum unless otherwise stated.

Details of generation of the germline NPY knockout mice were published previously (Karl et al., 2008).

Female wild type (WT) and NPY-/- mice (both on a (C57BL/6JAusb background) at 10 weeks of age were used to

investigate the effect of sucralose on food intake. Sucralose was administered to mice orally and voluntarily by

incorporating sucralose into synthetically flavoured jelly and mice were trained to eat the jelly as described previously

(Cox et al., 2010; Zhang et al., 2010)) Treatment jelly contained 7.5mg of sucralose per dose, and vehicle jelly contained

0.03 mg of sucralose to allow voluntary administration of the jelly. Jelly was given at 1630hr and mice typically consume

the entire piece of jelly (~290 µL) within 5 minutes. Food intake was determined using Promethion metabolic cages

(PromethionTM Line, Sable Systems International, NV USA). The experimental protocol included a 3-day run-in period

in which mice were acclimatized to the chambers while receiving control jelly. Jelly was given at 1630hr and food intake

was recorded continuously. Subsequently, mice were randomly assigned to either the control or treatment group which

received vehicle and treatment jelly, respectively, daily at 1630 hr and food intake was recorded over 7 days.

Statistical Analysis: Data are represented as means ± SEM. Statistical tests were performed use unpaired t test, One-way

ANOVA with Turkey’s multiple comparisons test, two-way ANOVA with Sidak's multiple comparisons test, or repeated

measures ANOVA as appropriate. All statistical analysis was performed using GraphPad Prism 6.0.

Supplemental References

Cole, S.H., Carney, G.E., McClung, C.A., Willard, S.S., Taylor, B.J., and Hirsh, J. (2005). Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J. Biol. Chem. 280, 14948-14955. Cox, H.M., Tough, I.R., Woolston, A.M., Zhang, L., Nguyen, A.D., Sainsbury, A., and Herzog, H. (2010). Peptide YY is critical for acylethanolamine receptor Gpr119-induced activation of gastrointestinal mucosal responses. Cell Metab. 11, 532-542. Dahanukar, A., Foster, K., van der Goes van Naters, W.M., and Carlson, J.R. (2001). A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila. Nat. Neurosci. 4, 1182-1186. Dahanukar, A., Lei, Y.T., Kwon, J.Y., and Carlson, J.R. (2007). Two Gr genes underlie sugar reception in Drosophila. Neuron 56, 503-516. Deshpande, S.A., Carvalho, G.B., Amador, A., Phillips, A.M., Hoxha, S., Lizotte, K.J., and Ja, W.W. (2014). Quantifying Drosophila food intake: comparative analysis of current methodology. Nat. Methods 11, 535-540. Dietzl, G., Chen, D., Schnorrer, F., Su, K.C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151-156. Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., and Birman, S. (2003). Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618-627. Haselton, A., Sharmin, E., Schrader, J., Sah, M., Poon, P., and Fridell, Y.W. (2010). Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance. Cell Cycle 9, 3063-3071.

Hiroi, M., Marion-Poll, F., and Tanimura, T. (2002). Differentiated response to sugars among labellar chemosensilla in Drosophila. Zoolog. Sci. 19, 1009-1018. Hodgson, E.S., Lettvin, J.Y., and Roeder, K.D. (1955). Physiology of a primary chemoreceptor unit. Science 122, 417-418. Inagaki, H.K., Ben-Tabou de-Leon, S., Wong, A.M., Jagadish, S., Ishimoto, H., Barnea, G., Kitamoto, T., Axel, R., and Anderson, D.J. (2012). Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583-595. Inagaki, H.K., Panse, K.M., and Anderson, D.J. (2014). Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila. Neuron 84, 806-820. Ishimoto, H., Wang, Z., Rao, Y., Wu, C.F., and Kitamoto, T. (2013). A novel role for ecdysone in Drosophila conditioned behavior: linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain. PLoS Genet. 9, e1003843. Ja, W.W., Carvalho, G.B., Mak, E.M., de la Rosa, N.N., Fang, A.Y., Liong, J.C., Brummel, T., and Benzer, S. (2007). Prandiology of Drosophila and the CAFE assay. Proc. Natl. Acad. Sci. U. S. A. 104, 8253-8256. Karl, T., Duffy, L., and Herzog, H. (2008). Behavioural profile of a new mouse model for NPY deficiency. Eur. J. Neurosci. 28, 173-180. Krashes, M.J., DasGupta, S., Vreede, A., White, B., Armstrong, J.D., and Waddell, S. (2009). A neural circuit mechanism integrating motivational state with memory expression in Drosophila. Cell 139, 416-427. Lee, H.G., Seong, C.S., Kim, Y.C., Davis, R.L., and Han, K.A. (2003). Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev. Biol. 264, 179-190. Masek, P., and Keene, A.C. (2013). Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons. PLoS Genet. 9, e1003710. Miyamoto, T., Slone, J., Song, X., and Amrein, H. (2012). A fructose receptor functions as a nutrient sensor in the Drosophila brain. Cell 151, 1113-1125. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., and Wold, B. (2008). Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621-628. Pfeiffenberger, C., Lear, B.C., Keegan, K.P., and Allada, R. (2010). Processing sleep data created with the Drosophila Activity Monitoring (DAM) System. Cold Spring Harb. Protoc. 2010, pdb prot5520. Pospisilik, J.A., Schramek, D., Schnidar, H., Cronin, S.J., Nehme, N.T., Zhang, X., Knauf, C., Cani, P.D., Aumayr, K., Todoric, J., et al. (2010). Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Cell 140, 148-160. Rulifson, E.J., Kim, S.K., and Nusse, R. (2002). Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296, 1118-1120. Sweeney, S.T., Broadie, K., Keane, J., Niemann, H., and O'Kane, C.J. (1995). Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341-351. Weiss, L.A., Dahanukar, A., Kwon, J.Y., Banerjee, D., and Carlson, J.R. (2011). The molecular and cellular basis of bitter taste in Drosophila. Neuron 69, 258-272. Wen, T., Parrish, C.A., Xu, D., Wu, Q., and Shen, P. (2005). Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity. Proc. Natl. Acad. Sci. U. S. A. 102, 2141-2146. Wu, Q., Wen, T., Lee, G., Park, J.H., Cai, H.N., and Shen, P. (2003). Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system. Neuron 39, 147-161.

Zhang, L., Lee, N.J., Nguyen, A.D., Enriquez, R.F., Riepler, S.J., Stehrer, B., Yulyaningsih, E., Lin, S., Shi, Y.C., Baldock, P.A., et al. (2010). Additive actions of the cannabinoid and neuropeptide Y systems on adiposity and lipid oxidation. Diabetes Obes. Metab. 12, 591-603.