Figures and data in Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila

Cite this article as: eLife 2020;9:e51595 doi: 10.7554/eLife.51595

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4 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 1 supplement

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Dome inhibition in adult muscle reduces lifespan, disrupts homeostasis, and causes AKT hyperactivation.

(A) STAT activity in different muscles in 10xSTAT92E-GFP reporter fly. One fly out of 5 shown. Upper panel: lateral view, Scale bar = 500 µm. Lower panels: dorsal thorax (left); dorsal abdomen (middle); tibia (right), Scale bar = 100 µm. (B) Lifespan of *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* at 29°C. Log-Rank test: χ² = 166, ***p<0.0001; Wilcoxon test: χ² = 157.7, ***p<0.0001. (C) Negative geotaxis assay of 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. Points represent mean height climbed in individual vials (~20 flies/vial), pooled from three independent experiments. Unpaired T-test: **p=0.0033. (D) Muscle (Phalloidin) and neutral lipid (LipidTox) of thorax samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. One representative fly per genotype is shown of six analysed. Scale bar = 50 µm. (E) Thin layer chromatography (TLC) of triglycerides in 7-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies, n = 3–4 per genotype. One experiment of two is shown. Unpaired T-Test: ***p<0.0001. (F) Glucose and trehalose (left) and glycogen (right) in 7-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies, pooled from two independent experiments. Unpaired T-Test (Glucose +Trehalose): ***p<0.0001 and unpaired T-Test (Glycogen): ***p<0.0001. (G) CO₂ produced, O₂ consumed, and RQ of 7-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. Box plots show data from one representative experiment of three, with data collected from a 24 hr measurement pooled from 3 to 4 tubes per genotype with 10 flies/tube. P values from Mann-Whitney test. (**H–L**) Western blots of leg protein from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. (H) Phospho-AKT (S505). One experiment of four is shown. Unpaired T-Test: ***p<0.0001. (I) Total AKT. One experiment of two is shown. Unpaired T-Test: **p=0.0017. (J) Phospho-p70 S6K (T398). One experiment of two is shown. Unpaired T-Test: ns p=0.0539. (K) Phospho-AMPKα (T173). One experiment of three is shown. Unpaired T-Test: ns p=0.1024. (L) Phospho-ERK (T202/Y204). One experiment of three is shown. Unpaired T-Test: ns p=0.0826.

Figure 1—figure supplement 1

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Further characterisation of the requirement for *dome* in adult muscle.

(A) STAT activity (*10xSTAT92E-GFP*) and muscle (*MHC-RFP*) colocalize in adult flies. One fly of 6 shown. Scale bar = 500 µm. (B) *dome* expression by qRT-PCR in thorax samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. Unpaired T-Test: *p=0.0102. (C) Lifespan of *UAS-dome^∆/+* and *24B-Gal80^ts > dome^∆* at 29°C. Log-Rank test: χ2 = 100.8, ***p<0.0001; Wilcoxon test: χ2 = 76.2, ***p<0.0001. (D) Lifespan of *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* at 25°C. Log-Rank test: χ2 = 61.83, ***p<0.0001; Wilcoxon test: χ2 = 55.18, ***p<0.0001. (E) Smurf assay of 14-day-old *24B-Gal80^ts/+* (n = 49) and *24B-Gal80^ts-dome^∆* flies (n = 18). Data pooled from two independent experiments. (F) Lifespan of *Mef2-Gal80^ts/+* and *Mef2-Gal80^ts > dome^∆* flies at 29°C. Log-Rank test: χ2 = 86.96, ***p<0.0001; Wilcoxon test: χ2 = 78.61, ***p<0.0001. (G) Negative geotaxis assay of 14-day-old *Mef2-Gal80^ts/+* and *Mef2-Gal80^ts > dome^∆* flies. Points represent mean climbing height of individual vials analysed (~20 flies/vial), pooled from three independent experiments. Unpaired T-Test: ***p<0.0001. (**H, I**) Western blots of protein from legs of 14-day-old *Mef2-Gal80^ts/+* and *Mef2-Gal80^ts > dome^∆* flies. One of three independent experiments is shown. (H) Phospho-AKT. Unpaired T-Test: *p=0.0145. (I) Total AKT. Unpaired T-Test: ***p<0.0003. (J) Western blots of Phospho-AKT in leg samples from 14-day-old *MHC-Gal4/+* and *MHC-Gal4 >dome^∆* (II) flies. One of two independent experiments is shown. Unpaired T-Test: ns p=0.2557. (K) Lifespan of *MHC-Gal4/+* and *MHC-Gal4 >dome^∆* (II) flies at 29°C. Log-Rank test: χ2 = 82.9, ***p<0.0001; Wilcoxon test: χ2 = 58.91, ***p<0.0001. (**L–R**) Expression by qRT-PCR of *Akt1* and insulin-like peptides in whole fly samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts-dome^∆* flies. All transcript levels are normalized to *Rpl1* and shown in arbitrary units [au]. P values in B, G, H-J, L-R from unpaired T-test. (S) Feeding assay of *24-Gal80^ts/+* and *24B2-Gal80^ts > dome^∆* flies at 29°C. n = 4 were analysed per genotype. Absorbance at 620 nm is shown in arbitrary units [au]. Unpaired T-Test: ns p=0.8819.

Figure 2 with 1 supplement

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Hop is required, but not sufficient, for Dome to control AKT.

(A) Lifespan of *w¹¹¹⁸* and *hop^Tum-L* flies at 29°C. Log-Rank test: χ² = 0.3223, ns p=0.5702; Wilcoxon test: χ² = 0.4756, ns p=0.4906. (B) Phospho-AKT in leg samples from 14-day-old *w¹¹¹⁸* and *hop^Tum-L* flies. One experiment of two is shown. Unpaired T-Test: ns p=0.6854. (C) Actin (Phalloidin) and neutral lipid (LipidTox) in flight muscle from 14-day-old *w¹¹¹⁸* and *hop^Tum-L* flies. One representative fly shown of six analysed per genotype. Scale bar = 50 µm. (D) Lifespan of *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, and *hop^Tum-L;24B-Gal80^ts > dome^∆* flies at 29°C. Log-Rank test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > dome^∆*): χ² = 319.4, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > dome^∆*): χ² = 280.2, ***p<0.0001. Log-Rank test (*24B-Gal80^ts/+* vs. *hop^Tum-L 24B-Gal80^ts > dome^∆*): χ² = 18.87, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts/+* vs. *hop^Tum-L 24B-Gal80^ts > dome^∆*): χ² = 20.83, ***p<0.0001. (E) Phospho-AKT in leg samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆* and *hop^Tum-L;24B-Gal80^ts > dome^∆* flies. P values from unpaired T-Test.

Figure 2—figure supplement 1

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Interactions of *dome* with AMPK, MAPK, and FOXO signalling in adult muscle.

(A) Phospho-AKT in leg samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > AMPKα-IR,* and *24B-Gal80^ts > AMPKβ-IR* flies. One of three independent experiments is shown. P values from unpaired T-Test. (B) Phospho-AKT in leg samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > rl* IR, and *24B-Gal80^ts > Dsor1* IR flies. One of three independent experiments is shown. P values from unpaired T-Test. (C) Lifespan of *24B-Gal80^ts/+*, *24B-Gal80^ts > rl* IR, and *24B-Gal80^ts > Dsor1* IR flies at 29°C. Log-Rank test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > rl* IR): χ2 = 60.29, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > rl* IR): χ2 = 58.32, ***p<0.0001; Log-Rank test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > Dsor1* IR): χ2 = 1.186, ns p=0.2760; Wilcoxon test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > Dsor1* IR): χ2 = 0.0033, ns p=0.9538. (D) Phospho-AKT in leg samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > hop* IR flies. One of two independent experiments is shown. Unpaired T-Test: *p=0.0187. (E) Lifespan of *24B-Gal80^ts/+* and *24B-Gal80^ts > hop* IR flies at 29°C. Log-Rank test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > hop* IR): χ2 = 546.4, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts/+* vs. *24B-Gal80^ts > hop* IR): χ2 = 458.1, ***p<0.0001. P values in A, C, E from unpaired T-test.

Figure 3 with 1 supplement

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AKT hyperactivation causes pathology in *24B-Gal80^ts > dome^∆* flies.

(A) Lifespan of *24B-Gal80^ts/+* and *24B-Gal80^ts > myr*-AKT at 29°C. Log-Rank test: χ² = 115.5, ***p<0.0001; Wilcoxon test: χ² = 123.6, ***p<0.0001. (B) Negative geotaxis assay of 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > myr*-AKT flies. Points represent mean height climbed in individual vials (~20 flies/vial), pooled from two independent experiments. Unpaired T-Test: *p=0.018. (C) TLC of triglycerides in 7-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > myr*-AKT flies, n = 3–4 per genotype. One experiment of two is shown. Unpaired T-Test: *p=0.0144. (D) Glucose and trehalose (left panel) and glycogen (right panel) in 7-day-old *24B-Gal80^ts/+* (n = 12) and *24B-Gal80^ts > myr*-AKT (n = 9) flies, pooled from two independent experiments. Unpaired T-Test (Glucose +Trehalose): ***p=0.0009 and unpaired T-Test (Glycogen): ***p<0.0001. (E) CO₂ produced, O₂ consumed, and RQ of 7-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > myr*-AKT flies. Box plots show data from one representative experiment of three, with data points collected from a 24 hr measurement pooled from 3 to 4 tubes per genotype with 10 flies/tube. P values from Mann-Whitney test. (F) Phalloidin and LipidTox staining of thorax samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > myr*-AKT flies. One representative fly per genotype is shown of 3 analysed per group in two independent experiments. Scale bar = 50 µm. (G) Lifespan of *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *UAS-dome^Δ/+, 24B-Gal80^ts > AKT-*IR, *UAS-AKT-IR/+*, *24B-Gal80^ts > AKT-IR;dome^∆* and UAS-*AKT-IR;dome^∆/+* flies at 29°C. Log-Rank test (*24B-Gal80^ts > dome^∆* vs. *24B-Gal80^ts > AKT-IR;dome^∆*): χ² = 101.0, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts > dome^∆* vs. *24B-Gal80^ts > AKT-IR;dome^∆*): χ² = 59.87, ***p<0.0001. (H) Lifespan of *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *foxo-GFP;24B-Gal80^ts/+*, and *foxo-GFP;24B-Gal80^ts > dome^∆* flies at 29°C. Log-Rank test (*24B-Gal80^ts > dome^∆* vs. *foxo-GFP;24B-Gal80^ts > dome^∆*): χ² = 114.0, ***p<0.0001; Wilcoxon test (*24B-Gal80^ts > dome^∆* vs. *foxo-GFP;24B-Gal80^ts > dome^∆*): χ² = 93.59, ***p<0.0001. (I) Glucose + trehalose and glycogen in 7-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^Δ*, *foxo-GFP;24B-Gal80/+*, and foxo-GFP; *24B-Gal80^ts > dome^Δ* flies. Statistical testing was performed with one-way ANOVA. (J) TLC of triglycerides in 7-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^Δ*, *foxo-GFP;24B-Gal80^ts/+*, and *foxo-GFP;24B-Gal80^ts > dome^Δ* flies. Statistical testing was performed with one-way ANOVA.

Figure 3—figure supplement 1

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Mutual regulation by AKT, Foxo, and Dome.

(A) *dome* expression by qRT-PCR in whole fly samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *24B-Gal80^ts > Akt*-IR, and *24B-Gal80^ts > Akt-IR;dome^∆* flies. (**B–F**) Gene xpression analysis of *dome, upd2, Pepck, Thor* and *InR* in whole fly samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *foxo-GFP;24B-Gal80^ts/+*, and *foxo-GFP;24B-Gal80^ts > dome^∆* flies. P values from unpaired T-test. (B) *dome*. (C) *upd2*. (D) *Pepck*. (E) *Thor*. (F) *InR*. (G) Western blot for GFP to detect the Foxo-GFP fusion protein in leg samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *foxo-GFP;24B-Gal80^ts/+*, and *foxo-GFP;24B-Gal80^ts > dome^∆* flies. (H) Western blot for Phospho-AKT in leg samples from 14-day-old *24B-Gal80^ts/+*, *24B-Gal80^ts > dome^∆*, *foxo-GFP;24B-Gal80^ts/+*, and *foxo-GFP;24B-Gal80^ts > dome^∆* flies. One of two independent experiments is shown. P values in A-D from unpaired T-test. (**I–M**) Gene expression analysis of Akh and six confirmed or putative Akh target genes in whole fly samples from 14-day-old *24B-Gal80^ts/+* and *24B-Gal80^ts > dome^∆* flies. Transcript levels are shown in arbitrary units [au]. P values from unpaired T-test. (I) *Akh*. (J) *Hsl*. (K) *bmm*. (L) *plin1*. (M) *plin2*.

Figure 4 with 2 supplements

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Plasmatocytes promote muscle Dome activity.

(A) Muscle (*MHC^YFP*) and plasmatocytes (*srpHemo-3xmCherry*) in 7-day-old flies. Plasmatocytes are found in close proximity to adult muscles. One representative fly of 5 is shown. Scale bar = 500 µm. (B) Legs and plasmatocytes in 7-day-old *10xSTAT92E-GFP;srpHemo-3xmCherry* flies. Muscle with high JAK-STAT activity (green) is surrounded by plasmatocytes (magenta). One representative fly of 5 is shown. Scale bar = 100 µm. (C) STAT activity and plasmatocytes in legs from control (*10xSTAT92E-GFP;crq-Gal4 >CD8-mCherry/+*) and *upd3*-overexpressing (*10xSTAT92E-GFP;crq-4>CD8mCherry/UAS-upd3*) flies. One representative fly of 10–14 is shown. Scale bar = 100 µm. Graph shows mean fluorescence intensity (MFI). Unpaired T-Test: ***p<0.0001. (D) STAT activity and plasmatocytes in legs from control (*10xSTAT92E-GFP;crq-Gal80^ts > CD8-mCherry/+*) and plasmatocyte-depleted (*10xSTAT92E-GFP;crq-Gal80^ts > CD8* mCherry/rpr) flies. One representative fly of six is shown. Scale bar = 250 µm. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (*10xSTAT92E-GFP;crq-Gal80^ts > CD8-mCherry/+*) and plasmatocyte-depleted (*10xSTAT92E-GFP;crq-Gal80^ts > CD8* mCherry/rpr flies). One representative experiment of three is shown. Graph shows STAT-GFP/α-tubulin for control (*crq-Gal80^ts/+*) and plasmatocyte-depleted (*crq-Gal80^ts > rpr*) leg samples. Unpaired T-Test: *p=0.0121. (F) Lifespan of *crq-Gal80^ts/+*, *crq-Gal80^ts > rpr*, *upd2^Δ upd3^Δ;crq-Gal80^ts/+*, and *upd2^Δ upd3^Δ;crq-Gal80^ts > rpr* flies at 29°C. Log-Rank test (*crq-Gal80^ts/+* vs. *crq-Gal80^ts > rpr*): χ2 = 101.7, ***p<0.0001; Wilcoxon test (*crq-Gal80^ts/+* vs. *crq-Gal80^ts > rpr*): χ2 = 107.8, ***p<0.0001; Log-Rank test (*crq-Gal80^ts/+* vs. *upd2 ^Δ upd3^Δ;crq-Gal80^ts > rpr*): χ2 = 60.03, ***p<0.0001; Wilcoxon test (*crq-Gal80^ts/+* vs. *upd2 ^Δ upd3^Δ;crq-Gal80^ts > rpr*): χ2 = 80.97, ***p<0.0001. (G) Actin (Phalloidin) and neutral lipid (LipidTox) in thorax samples from 14-day-old *crq-Gal80^ts/+*, *crq-Gal80^ts > rpr*, *upd2 ^Δ upd3^Δ;crq-Gal80^ts/+*, and *upd2 ^Δ upd3^Δ;crq-Gal80^ts > rpr* flies. One representative fly per genotype shown of 6 analysed per group. Scale bar = 50 µm. (H) Lifespan of *crq-Gal4/+* and *crq-Gal4 >upd1* IR flies at 29°C. Log-Rank test: χ2 = 31.36, ***p<0.0001; Wilcoxon test: χ2 = 22.17, ***p=0.0001. (I) Expression by qRT-PCR of *upd1*, *upd2* and *upd3* in thorax samples of *crq-Gal4/+* and *crq-Gal4 >upd1* IR flies, data from four independent samples of each genotype.. Unpaired T-Test (*upd1*): ns p=0.848, unpaired T-Test (*upd2*): *p=0.0449 and unpaired T-Test (*upd3*): **p=0.0038. (J) Lifespan of *crq-Gal4/+*, *upd2 ^Δ upd3^Δ;UAS-upd1-IR/+*, and *upd2^Δ upd3^Δ;crq-Gal4 >upd1* IR flies at 29°C. Log-Rank test (*crq-Gal4/+* vs. *upd2 ^Δ upd3^Δ;crq-Gal4 >upd1* IR): χ2 = 41.12, ***p<0.0001; Wilcoxon test (*crq-Gal4/+* vs. *upd2^Δ upd3^Δ;crq-Gal4 >upd1* IR): χ2 = 54.47, ***p<0.0001 Log-Rank test (*crq-Gal4/+* vs. *upd2 ^Δ upd3^Δ;UAS-upd1-IR/+*): χ2 = 14.46, ***p<0.0001; Wilcoxon test (*crq-Gal4/+* vs. *upd2^Δ upd3^Δ;UAS-upd1-IR/+*): χ2 = 19.99, ***p<0.0001. P values in C, E, H from unpaired T-test.

Figure 4—figure supplement 1

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Further characterisation of plasmatocyte-depleted flies.

(A) Plasmatocyte depletion in *crq-Gal80^ts > rpr* flies (right) and controls (left) at 24 hr, 48 hr and 72 hr at 29°C. One out of four flies is shown. Scale bar = 250 µm. (B) Quantification of mCherry-positive plasmatocytes in *crq-Gal80^ts > rpr* flies (right) and controls (left) by flow cytometry after 72 hr at 29°C. n = 3 per group was analysed. Unpaired T-Test: *p=0.0337. (C) Negative geotaxis assay of 14-day-old *crq-Gal80^ts/+*, *crq-Gal80^ts > rpr*, *upd2^∆upd3^∆;UAS-rpr/+* and *upd2^∆upd3^∆;crq-Gal80^ts > rpr* flies. Points represent mean climbing height of individual vials analysed (~20 flies/vial). Unpaired T-Test: *p=0.0194.

Figure 4—figure supplement 2

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Further characterisation of requirements for specific Upds.

(A) Glucose + trehalose and glycogen in 7-day-old *crq-Gal80^ts/+*, *crq-Gal80^ts > rpr*, *upd2^Δ upd3^Δ;crq-Gal80^ts/+*, and *upd2^Δ upd3^Δ; crq-Gal80^ts > rpr* flies. (B) TLC of triglyceride in 7-day-old *crq-Gal80^ts/+*, *crq-Gal80^ts > rpr*, *upd2^Δ upd3^Δ;crq-Gal80^ts/+*, and *upd2^Δ upd3^Δ;crq-Gal80^ts > rpr* flies, n = 2–3 samples per genotype. (C) Western blot analysis of STAT-driven GFP in legs from 7-day-old *w¹¹¹⁸*, *upd3^Δ*, *upd2^Δ*, and *upd2^Δ upd3^Δ* flies. One representative experiment of two is shown. (D) STAT activity and plasmatocytes in legs from 7-day-old control (*crq-Gal4/+*), *upd1* knockdown (*crq-Gal4 >upd1* IR), and *upd3* knockdown (*crq-Gal4 >upd3* IR) flies. One representative fly is shown of 5–7 imaged for each genotype. Scale bar = 100 µm. Mean fluorescence intensity (MFI) is shown for all flies imaged. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (*crq-Gal4/+*), *upd1* knockdown (*crq-Gal4 >upd1* IR), and *upd3* knockdown (*crq-Gal4 >upd3* IR) flies. One of two independent experiments is shown. (F) Western blot analysis of STAT-driven GFP in thorax from 7-day-old *crq-Gal4/+*, crq-Gal4 >*upd1*, *upd2^Δupd3^Δ;crq-Gal4/+* and *upd2^Δupd3^Δ;crq-Gal4 >upd1* IR flies. P values in A-F from unpaired T-test.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. melanogaster)	w¹¹¹⁸; tubulin-Gal80^ts/SM6a;24B-Gal4/TM6c, Sb¹	This study		Inserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GawB]how24B
Genetic reagent (D. melanogaster)	w¹¹¹⁸; tubulin-Gal80^ts/SM6a;Mef2-Gal4/TM6c, Sb¹	This study		Inserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GAL4-Mef2.R]3
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;UAS-dome^∆/TM6c, Sb¹	Brown et al., 2001		Gift of James Castelli-Gair Hombría
Genetic reagent (D. melanogaster)	w¹¹¹⁸;UAS-dome^∆/CyO	Brown et al., 2001		Gift of James Castelli-Gair Hombría
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;UAS-myr-AKT/TM6c, Sb¹	Stocker et al., 2002		Gift of Ernst Hafen
Genetic reagent (D. melanogaster)	w;UAS-AMPKα-IR	Vienna Drosophila Research Center (VDRC)	RRID:FlyBase_FBst0478025; VDRC 106200
Genetic reagent (D. melanogaster)	w;UAS-AMPKβ-IR	VDRC	RRID:FlyBase_FBst0476347; VDRC 104489
Genetic reagent (D. melanogaster)	w;UAS-rl-IR	VDRC	RRID:FlyBase_FBst0480887; VDRC 109108
Genetic reagent (D. melanogaster)	w;UAS-Dsor1-IR	VDRC	RRID:FlyBase_FBst0479098; VDRC 107276
Genetic reagent (D. melanogaster)	w¹¹¹⁸;foxo^GFP	BDSC	RRID:BDSC_38644	Inserted Element: PBac[foxo-GFP.FLAG]VK00037
Genetic reagent (D. melanogaster)	w;UAS-AKT-IR	VDRC	RRID:FlyBase_FBst0475561; VDRC 103703
Genetic reagent (D. melanogaster)	w¹¹¹⁸;10xSTAT92E-GFP	BDSC Bach et al., 2007	RRID:BDSC_26197	Inserted Element: P[10XStat92E-GFP]1
Genetic reagent (D. melanogaster)	w¹¹¹⁸;MHC-Gal4,MHC-RFP/SM6a	BDSC	RRID:BDSC_38464	Inserted Element: P[Mhc-GAL4.F3-580]2; P[Mhc-RFP.F3-580]2
Genetic reagent (D. melanogaster)	w upd2 ^Δ upd3^Δ;;;	BDSC	RRID:BDSC_55729
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;crq-Gal4/TM6 c, Sb¹			Gift of Nathalie Franc
Genetic reagent (D. melanogaster)	w¹¹¹⁸;tub-Gal80^ts;TM2/TM6 c, Sb¹	BDSC	RRID:BDSC_7108
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;UAS-rpr/TM6 c, Sb¹	BDSC	RRID:BDSC_5824
Genetic reagent (D. melanogaster)	w¹¹¹⁸;UAS-CD8-mCherry	BDSC	RRID:BDSC_27391
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;srpHemo-3xmCherry/TM6c, Sb¹	Gyoergy et al., 2018
Genetic reagent (D. melanogaster)	w;UAS-hop-IR	VDRC	RRID:FlyBase_FBst0463355; VDRC 40037
Genetic reagent (D. melanogaster)	w;UAS-upd1-IR/SM6a	VDRC	RRID:FlyBase_FBst0459787; VDRC 3282
Genetic reagent (D. melanogaster)	w;UAS-upd3-IR	VDRC	RRID:FlyBase_FBst0456774; VDRC 27134
Genetic reagent (D. melanogaster)	w¹¹¹⁸;;UAS-upd3/TM6 c, Sb¹			Gift of Bruce Edgar
Genetic reagent (D. melanogaster)	w¹¹¹⁸;UAS-2xeGFP/SM6a	BDSC	RRID:BDSC_6874
Genetic reagent (D. melanogaster)	w¹¹¹⁸ hop^Tum-L/FM7h	BDSC	RRID:BDSC_8492	backcrossed onto w¹¹¹⁸ background
Sequence-based reagent	Akt1_forward	This study	PCR primers	5’-ctttgcgagtattaactggacaga-3’
Sequence-based reagent	Akt1_reverse	This study	PCR primers	5’-ggatgtcacctgaggcttg-3’
Sequence-based reagent	Ilp2_forward	This study	PCR primers	5’-atcccgtgattccaccacaag-3’
Sequence-based reagent	Ilp2_reverse	This study	PCR primers	5’-gcggttccgatatcgagtta-3’
Sequence-based reagent	Ilp3_forward	This study	PCR primers	5’-caacgcaatgaccaagagaa-3’
Sequence-based reagent	Ilp3_reverse	This study	PCR primers	5’-tgagcatctgaaccgaact-3’
Sequence-based reagent	Ilp4_forward	This paper	PCR primers	5’-gagcctgattagactgggactg-3’
Sequence-based reagent	Ilp4_reverse	This paper	PCR primers	5’-tggaccggctgcagtaac-3’
Sequence-based reagent	Ilp5_forward	This paper	PCR primers	5’-gccttgatggacatgctga-3’
Sequence-based reagent	Ilp5_reverse	This paper	PCR primers	5’-agctatccaaatccgcca-3’
Sequence-based reagent	Ilp6_forward	This paper	PCR primers	5’-cccttggcgatgtatttcc-3’
Sequence-based reagent	Ilp6_reverse	This paper	PCR primers	5’-cacaaatcggttacgttctgc-3’
Sequence-based reagent	Ilp7_forward	This paper	PCR primers	5’-cacaccgaggagggtctc-3’
Sequence-based reagent	Ilp7_reverse	This paper	PCR primers	5’-caatatagctggcggacca-3’
Sequence-based reagent	dome_forward	This paper	PCR primers	5’-cggactttcggtactccatc-3’
Sequence-based reagent	dome_reverse	This paper	PCR primers	5’-accttgatgaggccaggat-3’
Sequence-based reagent	upd1_forward	This paper	PCR primers	5’-gcacactgatttcgatacgg-3’
Sequence-based reagent	upd1_reverse	This paper	PCR primers	5’- ctgccgtggtgctgtttt −3’
Sequence-based reagent	upd2_forward	This paper	PCR primers	5’-cggaacatcacgatgagcgaat-3’
Sequence-based reagent	upd2_reverse	This paper	PCR primers	5’-tcggcaggaacttgtactcg-3’
Sequence-based reagent	upd3_forward	This paper	PCR primers	5’-actgggagaacacctgcaat-3’
Sequence-based reagent	upd3_reverse	This paper	PCR primers	5’-gcccgtttggttctgtagat-3’
Sequence-based reagent	Pepck_forward	This paper	PCR primers	5’-ggataaggtggacgtgaag-3’
Sequence-based reagent	Pepck_reverse	This paper	PCR primers	5’-acctcctgcgaccagaact-3’
Sequence-based reagent	Thor_forward	This paper	PCR primers	5’-caggaaggttgtcatctcgga-3’
Sequence-based reagent	Thor_reverse	This paper	PCR primers	5’-ggagtggtggagtagagggtt-3’
Sequence-based reagent	InR_forward	This paper	PCR primers	5'-gcaccattataaccggaacc-3'
Sequence-based reagent	InR_reverse	This paper	PCR primers	5'-ttaattcatccatgacgtgagc-3'
Sequence-based reagent	Akh_forward	This paper	PCR primers	5’- agccgtgctcttcatgct-3’
Sequence-based reagent	Akh_reverse	This paper	PCR primers	5’-aaaggttccaggaccagctc-3’
Sequence-based reagent	Hsl_forward	This paper	PCR primers	5’-cttggaaatacttgaggggttg-3’
Sequence-based reagent	Hsl_reverse	This paper	PCR primers	5’-agatttgatgcagttctttgagc-3’
Sequence-based reagent	bmm_forward	This paper	PCR primers	5’-gtctcctctgcgatttgccat-3’
Sequence-based reagent	bmm_reverse	This paper	PCR primers	5’-ctgaagggacccagggagta-3’
Sequence-based reagent	plin1_forward	This paper	PCR primers	5’-gcgttctatggtagccttcag-3’
Sequence-based reagent	plin1_reverse	This paper	PCR primers	5’-gcgtccggatagaaagctg-3’
Sequence-based reagent	plin2_forward	This paper	PCR primers	5’-gcagaatggcaagagttctga-3’
Sequence-based reagent	plin2_reverse	This paper	PCR primers	5’-actgtgtgtaggactggatcctc-3’
Sequence-based reagent	Rpl1_forward	This paper	PCR primers	5’-tccaccttgaagaagggcta-3’
Sequence-based reagent	Rpl1_reverse	This paper	PCR primers	5’-ttgcggatctcctcagactt-3’
Peptide, recombinant protein	Trehalase	Sigma Aldrich	T8778
Peptide, recombinant protein	β-Amyloglucosidase	Sigma Aldrich	10115
Antibody	anti-phospho(Ser505)-AKT	Cell Signal Technology (CST)	Cat# 4054; RRID:AB_331414	WB (1:1000)
Antibody	anti-AKT	Cell Signal Technology (CST)	Cat# 4691; RRID:AB_915783	WB (1:1000)
Antibody	anti-phospho(Thr172)-AMPKα	Cell Signal Technology (CST)	Cat# 2535; RRID:AB_331250	WB (1:1000)
Antibody	anti-phospho(Thr389)-p70 S6 kinase	Cell Signal Technology (CST)	Cat# 9206; RRID:AB_2285392	WB (1:1000)
Antibody	anti-GFP	Cell Signal Technology (CST)	Cat# 2956; RRID:AB_1196615	WB (1:1000)
Antibody	anti-phospho-p44/42 MAPK (Erk1/2)	Cell Signal Technology (CST)	Cat# 4370; RRID:AB_2315112	WB (1:1000)
Antibody	anti-α-tubulin	Developmental Studies Hybridoma Bank)	Clone 12G10; RRID:AB_1157911	WB (1:3000)
Antibody	HRP anti-rabbit IgG	Cell Signal Technology (CST)	Cat# 7074; RRID:AB_2099233	WB (1:5000)
Antibody	HRP anti-mouse IgG	Cell Signal Technology (CST)	Cat# 7076; RRID:AB_330924	WB (1:5000)
Commercial assay or kit	First Strand cDNA Synthesis Kit	Thermo Scientific	K1622
Commercial assay or kit	Sensimix SYBR Green no-ROX	Bioline	QT650-05
Chemical compound, drug	Bromophenol blue	Sigma Aldrich	SML1656
Chemical compound, drug	Xylene cyanol	Carl Roth	A513.1
Chemical compound, drug	Brilliant Blue FCF	Sigma Aldrich	80717
Other	HCS Lipid Tox Deep Red	Thermo Fisher	H34477	IF (1:200)
Other	Alexa Fluor 488 Phalloidin	Thermo Fisher	A12379	IF (1:20)
Other	Fluoromount-G	ebioscience	00-4958-02
Other	TRIzol	Invitrogen	AM9738
Other	Fixable Viability Dye 780	ebioscience	65-0865-18	FC (1:1000)
Other	Supersignal West Pico Chemiluminescent Substrate	Thermo Scientific	34077
Other	Supersignal West Femto Chemiluminescent Substrate	Thermo Scientific	34094
Other	Glucose Reagent	Sentinel Diagnostics	17630H
Software, algorithm	ImageJ	ImageJ	RRID:SCR_003070
Software, algorithm	GraphPad Prism	GraphPad	RRID:SCR_002798
Software, algorithm	FlowJo	FlowJo	RRID:SCR_008520)