High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila
- Center for Neurointelligence, School of Medicine, Chongqing University & Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College, Chongqing University, China
- Shenzhen Bay Laboratory, China
- Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, China
Figures
Figure 1 with 5 supplements
HFD promotesstarvation-induced hyperactivity in adult Drosophila.
(A–B) Wild-type Oregon-R virgin female flies fed with normal fly food (A, ND, blue) or high-fat diet (B, HFD, orange) were assayed in the presence (dark color) and absence (light color) of 5% …
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Figure 1—source data 1
Raw data of the behavioral experiments shown in Figure 1 and Figure 1—figure supplements 1–5.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Starvation-induced hyperactivity across different wild-type fly strains.
(A1, B1, C1, D1) Midline crossing activity of different wild-type strains assayed in the presence or absence of 5% sucrose (n = 46–63). (A2, B2, C2, D2) Average daily midline crossing activity in …
Figure 1—figure supplement 2
HFD promotes starvation-induced hyperactivity in a dose-dependent manner.
Wild-type Oregon-R virgin female flies fed with normal fly food (ND, blue) or different concentrations of coconut oil (orange) were assayed in the presence (dark color) and absence (light color) of …
Figure 1—figure supplement 3
HFD feeding enhanced starvation-induced food seeking.
(A) Schematic diagram of the video recording-based food seeking assay. Briefly, individual flies starved for 24 hr were introduced into a behavioral chamber with a small food patch (5% sucrose) …
Figure 1—figure supplement 4
Dietary saturated fat promotes starvation-induced hyperactivity.
Wild-type Oregon-R virgin female flies fed with normal fly food (ND, blue) or different types of lipids (orange) were assayed in the presence (dark color) and absence (light color) of 5% sucrose …
Figure 1—figure supplement 5
Dietary sugar does not affect starvation-induced hyperactivity.
Wild-type Oregon-R virgin female flies fed with ND (blue) or HSD (orange) were assayed in the presence (dark color) and absence (light color) of 5% sucrose using DAMS-based locomotion assay (n = 41–6…
Figure 2 with 1 supplement
HFD increases neuronal AKHR protein.
(A–B) AKH and DILP2 mRNA levels of wild-type virgin female flies fed with ND or HFD. The fly heads and associated tissues were collected and subjected to RNAseq (A) or quantitative RT-PCR (B) (n = 3 …
Figure 2—figure supplement 1
HFD increases neuronal AKHR protein accumulation in both fed and starved conditions.
(A-B) AKHR-flag knock-in flies were fed with ND or HFD for 5 d, and then starved for 18 hr or not. AKHR-flag protein level in the head tissue was analyzed by western blot (A, n = 3 biological …
Figure 3
AKHR accumulation is induced by the suppression of autophagy.
(A–B) Quantification of AKHR protein levels in cultured Drosophila S2 cells upon the treatment of proteasome and lysosome inhibitors (n = 3 biological replicates). Cultured cells transiently …
Figure 4 with 4 supplements
Inhibition of autophagy increases starvation-induced hyperactivity.
(A–B) Representative traces (A) and quantification (B) of peak calcium responses of AKHR+ neurons to 316 nM AKH upon suppressing neuronal autophagy in AKHR+ neurons (n = 7–10). (C–D) Midline …
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Figure 4—source data 1
Raw data of the behavioral experiments shown in Figure 4 and Figure 4—figure supplements 1–4.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
AKHR gene is required for the enhancement of starvation-induced hyperactivity by HFD.
(A–B) Midline crossing activity of ND- and HFD-fed AKHR-/- flies assayed in the presence or absence of 5% sucrose (n = 43–73). (C–D) Average daily midline crossing activity of fed flies (C) and …
Figure 4—figure supplement 2
Silencing AKHR+ neurons diminishes the effect of HFD feeding to enhance starvation-induced hyperactivity.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 39–64). (C) Average daily midline crossing activity of fed flies …
Figure 4—figure supplement 3
Activation AKHR+ neurons enhances starvation-induced hyperactivity under ND feeding.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 30–61). Red line indicates the time window for quantifying average …
Figure 4—figure supplement 4
Knocking down ATG7 in AKHR+ neurons promotes starvation-induced hyperactivity.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 30–60). (C) Average daily midline crossing activity of fed flies …
Figure 5 with 2 supplements
HFD activates TOR signaling.
(A–D) p70s6k/AKT/AMPK phosphorylation under ND vs. HFD feeding conditions. The head tissues of wild-type flies fed with ND or HFD were harvested and subjected to western blot with phosphorylated …
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Figure 5—source data 1
Top differentially expressed genes upon HFD feeding.
Wild-type virgin female flies were fed with ND or HFD for 5 d before their head tissues harvested and subjected to RNAseq. Top 40 differentially expressed genes (20 up-regulated and 20 down-regulated) were shown.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
KEGG pathway analysis of differentially expressed genes upon HFD feeding.
Wild-type virgin female flies were fed with ND or HFD for 5 d before their head tissues harvested and subjected to RNAseq. The highly influenced KEGG pathways were shown (red: up-regulated by HFD; …
Figure 5—figure supplement 2
TOR downstream genes are modulated by HFD feeding.
Heat map of differentially expressed genes enriched in TOR signaling pathways. Wild-type virgin female flies were fed with ND or HFD for 5 d before their head tissues harvested and subjected to …
Figure 6 with 3 supplements
AMPK-TOR signaling modulates starvation-induced hyperactivity.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 35–58). (C) Average daily midline crossing activity of fed flies …
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Figure 6—source data 1
Raw data of the behavioral experiments shown in Figure 6 and Figure 6—figure supplements 1–3.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Rapamycin feeding suppresses starvation-induced hyperactivity.
Wild-type virgin female flies fed with ND (blue) or ND plus rapmycin (green) were assayed in the presence (dark color) and absence (light color) of 5% sucrose using DAMS-based locomotion assay (n = 4…
Figure 6—figure supplement 2
Knocking down TSC1 in AKHR+ neurons promotes starvation-induced hyperactivity.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 36–46). (C) Average daily midline crossing activity of fed flies …
Figure 6—figure supplement 3
Suppressing AMPK signaling in AKHR+ neurons promotesstarvation-induced hyperactivity.
(A–B) Midline crossing activity of indicated genotypes and diet treatments assayed in the presence or absence of 5% sucrose (n = 30–59). (C) Average daily midline crossing activity of fed flies …
Figure 7 with 1 supplement
LpR1 is required for HFD-strengthened hyperactivity under starvation.
(A–B) LTP protein levels in the hemolymph. Hemolymph was collected from flies fed with ND or HFD and subjected to LC-MS/MS analysis. Volcano plot (A) shows peptides with differential enrichment …
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Figure 7—source data 1
Raw data of the behavioral experiments and mass spec experiments shown in Figure 7.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig7-data1-v2.xlsx
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Figure 7—source data 2
Top differentially expressed peptides upon HFD feeding.
Wild-type virgin female flies were fed with ND or HFD for 5 d before their hemolymph harvested and subjected to LC-MS/MS. Top 40 differentially expressed peptides (20 up-regulated and 20 down-regulated) were shown.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
KEGG pathway analysis of differentially expressed peptides upon HFD feeding.
Wild-type virgin female flies were fed with ND or HFD for 5 d before their hemolymph harvested and subjected to LC-MS/MS. The highly influenced KEGG pathways were shown (red: up-regulated by HFD; …
Figure 8
Diet-independent hyperlipidemia promotes the excitability of AKHR+ neurons and starvation-induced hyperactivity.
(A–B) Average levels of cholesterol (A) and triglyceride (B) in the hemolymph of the indicated genotypes (n = 4–11 biological replicates, each containing 60 flies). (C–F) The levels of …
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Figure 8—source data 1
Raw data of the behavioral experiments shown in Figure 8.
- https://cdn.elifesciences.org/articles/53103/elife-53103-fig8-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | UAS-TORTED | Bloomington Drosophila Stock Center | Cat. #7013; RRID:BDSC_7013 | FlyBase symbol: P{w+mC=UAS-Tor.TED} |
| Genetic reagent (D. melanogaster) | UAS-AMPK-DN | Bloomington Drosophila Stock Center | Cat. #32112; RRID:BDSC_32112 | FlyBase symbol: P{w+mC=UAS-AMPKalpha.K57A} |
| Genetic reagent (D. melanogaster) | UAS-mCD8GFP | Bloomington Drosophila Stock Center | Cat. #32186; RRID:BDSC_32186 | FlyBase symbol: P{y+t7.7 w+mC = 10XUAS-IVS-mCD8::GFP} |
| Genetic reagent (D. melanogaster) | UAS-mCD8GFP | Bloomington Drosophila Stock Center | Cat. #32186; RRID:BDSC_32186 | FlyBase symbol: P{y+t7.7 w+mC = 10XUAS-IVS-mCD8::GFP} |
| Cell line (include species here) | ppl-GAL4 | Bloomington Drosophila Stock Center | Cat. #58768; RRID:BDSC_58768 | FlyBase symbol: P{w+mC = ppl-GAL4.P} |
| Genetic reagent (D. melanogaster) | UAS-NaChBac | Bloomington Drosophila Stock Center | Cat. # 9469; RRID:BDSC_9469 | FlyBase symbol: P{w+mC = UAS-NaChBac} |
| Genetic reagent (D. melanogaster) | UAS-nSyb-AD-AKHR-BD | Our laboratory PMID:27612383 | Dr. Liming Wang (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | UAS-nSyb-AD | Our laboratory PMID:27612383 | Dr. Liming Wang (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | UAS- AKHR-BD | Our laboratory PMID:27612383 | Dr. Liming Wang (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | UAS- AKHR-/- | Our laboratory PMID:27612383 | Dr. Liming Wang (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | UAS- kir2.1 | Our laboratory PMID:30209352 | Dr. Liming Wang (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | UAS-TSC1 RNAi | Tsinghua Fly Center | Cat. #5074 | |
| Genetic reagent (D. melanogaster) | UAS-ATG7 RNAi | Tsinghua Fly Center | Cat. #2793 | |
| Genetic reagent (D. melanogaster) | UAS-LpR1 RNAi | Tsinghua Fly Center | Cat. #2568 | |
| Genetic reagent (D. melanogaster) | UAS-ATG5 RNAi | Laboratory of Dr. Chao Tong | Dr. Chao Tong (Zhejiang University) | |
| Genetic reagent (D. melanogaster) | AKHR-FLAG | This paper | Dr. Liming Wang (Zhejiang University) | |
| Cell line (D. melanogaster) | S2 | Thermofisher | Cat. #R69007 | |
| Antibody | Anti-phospho-Akt (Ser473) (Rabbit polyclonal) | Cell Signaling Technology | Cat. #9271; RRID:AB_329825 | WB (1:1000) |
| Antibody | Anti-phospho-AMPK (Thr172) (Rabbit polyclonal) | Cell Signaling Technology | Cat. #2535; RRID:AB_331250 | WB (1:1000) |
| Antibody | Anti-GFP (Rabbit polyclonal) | Cell Signaling Technology | Cat. #2555; RRID:AB_10692764 | WB (1:1000) |
| Antibody | Anti-FLAG (Rabbit polyclonal) | Cell Signaling Technology | Cat. #86861; RRID:AB_2800094 | WB (1:1000) |
| Antibody | Anti-HA (Rabbit polyclonal) | Cell Signaling Technology | Cat. #3724; RRID:AB_1549585 | WB (1:1000) |
| Antibody | Anti-HA (Mouse monoclonal) | Cell Signaling Technology | Cat. #2367, RRID:AB_10691311 | IF(1:200); WB (1:1000) |
| Antibody | Anti-actin (Rabbit polyclonal) | Sigma-Aldrich | Cat. #A5316; RRID:AB_476743 | WB (1:5000) |
| Antibody | Anti-AKH (Rabbit polyclonal) | biorbyt | Cat. #orb97730 | DOT BLOT (1:1000) |
| Antibody | Anti-Dilp2 (Rabbit polyclonal) | Laboratory of Dr. Zhefeng Gong | DOT BLOT (1:1000) Dr. Zhefeng Gong (Zhejiang Universtiy) | |
| Recombinant DNA reagent | AKHR-HA (plasmid) | This paper | Dr. Liming Wang (Zhejiang University) | |
| Recombinant DNA reagent | GFP-ATG8 (plasmid) | This paper | Dr. Liming Wang (Zhejiang University) | |
| Peptide, recombinant protein | AKH | PMID:2117437 | pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 | |
| Chemical compound, drug | MG132 | Beyotime Biotechnology | Cat. #S1748 | |
| Chemical compound, drug | AICAR | Beyotime Biotechnology | Cat. #S1516 | |
| Chemical compound, drug | MHY (MHY1485) | Sigma-Aldrich | Cat. #SML0810 | |
| Chemical compound, drug | dorsomorphin | Sigma-Aldrich | Cat. #P5499 | |
| Chemical compound, drug | Chloroquine (CQ) | Sigma-Aldrich | Cat. #C6628 | |
| Chemical compound, drug | Rapamycin | Sangon Biotech | Cat. #A606203 | |
| Software, algorithm | GraphPad Prism 6 | GraphPad Software | www.graphpad.com |
