1. Volume 23, Issue 4, Pages 675-684 (April 2016)
The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response 
Neha Agrawal, Renald Delanoue, Alessandra Mauri, Davide Basco, Matthieu Pasco, Bernard Thorens, Pierre Léopold 
Cell Metabolism 
Volume 23, Issue 4, Pages (April 2016)
DOI: /j.cmet
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2. Cell Metabolism 2016 23, 675-684DOI: (10.1016/j.cmet.2016.03.003)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Eiger/ TNF-α and the Nutritional Regulation of Body Size
(A and B) Reducing egr expression in the fat body with the lpp-GAL4 line with two different RNAi lines increases pupal volume compared to controls (lpp-GAL4 or RNAi lines crossed with wild-type flies) in low-protein diet (LPD), but not in control (Ctrl) conditions. Expression of the extracellular form of Egr lacking the transmembrane domain with the lpp-GAL4 decreases pupal volume compared to controls in both LPD and control conditions. Statistically significant percentage changes in pupal volume are indicated above the genotypes compared with lpp-GAL4 crossed with wild-type flies.
(C) An egr-GAL4 enhancer trap line is strongly expressed in the fat body as visualized by nls-GFP (green). Nuclei are labeled with DAPI (blue). The bottom panel shows the specificity of egr-GAL4 expression that is restricted to fat body cells but not the salivary gland (indicated by SG). Scale bar, 50 μm. Error bars represent SEM; ∗p < 0.05 and ∗∗p < 0.01 versus control. See also Figure S1.
Cell Metabolism  , DOI: ( /j.cmet )
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4. Figure 2
Nutritional Control of Egr Release through TNF-α Converting Enzyme
(A–D, G, and H) Measurement of egr or TACE transcript levels in dissected fat body by quantitative RT-PCR. Fold changes are indicated with rp-49 used as an internal reference; error bars represent SEM.
(A) egr transcript levels are unchanged in fat body dissected from larvae reared on LPD compared to control diet.
(B) TACE transcript levels are significantly increased in fat body dissected from larvae reared on LPD as compared to control diet.
(C and D) TACE transcript levels are unchanged in fat body dissected from larvae subjected to a short 4 hr starvation but significantly elevated on a long 18 hr starvation.
(E and F) Reducing TACE expression in the fat body with the lpp-GAL4 increases pupal volume compared to lpp-GAL4 crossed with wild-type flies in LPD but not in control food conditions.
(G) TACE transcript levels are unchanged in fat body dissected from larvae reared on a low-sugar diet as compared to control diet.
(H) TACE transcript levels are significantly increased in the fat body by reducing expression of the amino acid transporter Slimfast (lpp > slifAnti; Colombani et al., 2003), reducing levels of raptor or overexpression of the TOR inhibitors TSC1 and TSC2 using lpp-GAL4.
Error bars represent SEM; ∗p < 0.05 and ∗∗p < 0.01 versus control. See also Figure S2.
Cell Metabolism  , DOI: ( /j.cmet )
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5. Figure 3 Humoral Eiger Is Regulated by Nutrition and TACE
(A) Hemolymph (Hemo) or whole-larval (WL) extracts from egr mutant or control larvae immunoblotted for endogenous Egr. The full-length (FL-Egr) and secreted forms of Egr (s-Egr) are clearly detected in the control conditions but are absent in the egr mutants. The levels of tubulin are used as a loading control.
(B) Reducing egr or TACE expression in the fat body with the lpp-GAL4 dramatically reduces secreted Egr levels in the hemolymph as compared to controls. The levels of Cv-d are used as a loading control.
(C) Secreted Egr levels are higher in the hemolymph from larvae subjected to amino acid starvation as compared to non-starved larvae. The levels of Cv-d are used as a loading control.
Cell Metabolism  , DOI: ( /j.cmet )
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6. Figure 4 JNK Signaling in the Larval IPCs
(A) JNK signaling is active in insulin-producing cells (IPCs) in larval brains carrying the JNK responsive pucE69 nuclear β-galactosidase reporter. Anti-Dilp2 is in red highlighting the IPCs and anti-βGAL is in green. Scale bar, 10 μm.
(B and C) Overexpressing puc with the dilp2-GAL4 driver increases pupal volume compared to controls (dilp2-GAL4 or RNAi lines crossed with wild-type flies) in LPD but not in control food conditions.
(D and E) Reducing egr expression in the fat body with the lpp-GAL4 increases dilp2 and dilp5 transcript levels in dissected larval brains in LPD but not in control food conditions. dilp3 levels remain unchanged in both food conditions.
Error bars represent SEM; ∗p < 0.05 and ∗∗p < 0.01 versus control. See also Figure S3.
Cell Metabolism  , DOI: ( /j.cmet )
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7. Figure 5 Grnd Is Expressed in the IPCs and Regulates Body Size
(A) Larval brains stained exclusively with the Anti-Grnd antibody show Grnd expression in IPCs of larval brain of wild-type larvae.
(B) Grnd expression overlaps with IPCs as visualized by nlsGFP (green) expressed with the dilp2-GAL4 driver confirming that Grnd is specifically localized to the IPCs. All scans were acquired sequentially to prevent bleed through during acquisition. Scale bar, 20 μm.
(C and D) Changes in pupal volume on reducing Grnd levels or expression of the full-length or activated form of Grnd in the IPCs with the dilp2-GAL4 driver compared to controls in LPD and control food conditions. Reducing grnd expression with the dilp2-GAL4 line increases pupal volume compared to controls (dilp2-GAL4 or RNAi line crossed with wild-type flies) in LPD but not in control conditions. Expression of the full-length Grnd with the dilp2-GAL4 decreases pupal volume compared to controls in LPD but not in control food conditions. Expression of the activated form of Grnd with the dilp2-GAL4 decreases pupal volume compared to controls in both LPD and control food conditions. Statistically significant percentage changes in pupal volume are indicated above the genotypes compared with dilp2-GAL4 crossed with wild-type flies. Error bars represent SEM; ∗p < 0.05 and ∗∗p < 0.01 versus control.
(E) Ex vivo culture experiments with wild-type brains or brains expressing full-length grnd (dilp2 > grnd) in the IPCs or dilp2 > grnd-RNAi incubated with hemolymph from larvae expressing venus-tagged Egr (Egr-venus) from the fat body. Egr-venus staining (in green) can be detected on the IPCs in wild-type and strongly in dilp2 > grnd brains, but not in dilp2 > grnd-RNAi brains (E7). Grnd staining in the three conditions is in red. All scans were acquired sequentially to prevent bleed through during acquisition. Scale bar, 10 μm. See also Figure S4.
Cell Metabolism  , DOI: ( /j.cmet )
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8. Figure 6 Functional Conservation of TNF Signaling
(A and B) TNF-α inhibits insulin expression in MIN6 cells and mouse pancreatic islets. The insulinoma-derived MIN6 cells (A) and isolated mouse islets (B) present a reduction of expression for both INS1 and INS2 genes after treatment with TNF-α (72hr) while the levels of control genes GAPDH and GUSB are not affected. The levels of β-actin (ACTb) expression are used for normalization. See Experimental Procedures for details.
(C) Fly adipose TNF-α signaling controls glycemia in high-sugar diet (HSD). The knockdown of grnd or traf1/4 in fat body cells significantly rescues the elevated glycemia in animals fed a HSD.
(D and E) The knockdown of grnd in fat body cells (lpp > grnd-RNAi) reduces InR transcript levels (used as a readout for insulin signaling) in the fat body of animals fed a HSD but not a control diet. Error bars represent SEM; ∗p < 0.05 and ∗∗∗p < versus control. See also Figure S5.
Cell Metabolism  , DOI: ( /j.cmet )
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