Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor by Renald Delanoue, Eleonora Meschi, Neha Agrawal, Alessandra Mauri, Yonit Tsatskis, Helen McNeill, and Pierre Léopold Science Volume 353(6307):1553-1556 September 30, 2016 Published by AAAS

Fig. 1 Mth and Sun are required for systemic growth. Mth and Sun are required for systemic growth. (A) Three different mth RNAi constructs (KK 102303, TRIP 36823, and BA3) decrease pupal size when driven in the IPC (dilp2>) (n > 20). (B) Silencing mth in the IPC induces accumulation of Dilp2 (n > 20). Fluorescence intensity measured as fold change (f.c.). (C) Silencing sun in the fat body (lpp-Gal4>sun-Ri) reduces pupal volume. No defect is observed when silencing in other tissues (myo1d-Gal4, gut; MHC-Gal4, muscle; elav-Gal4, CNS; or dilp2-Gal4, IPC). (D) sun silencing in the fat body causes Dilp2 accumulation in the IPC (n > 60). (E) sun overexpression in the fat body partially rescues the pupal size reduction observed in larvae fed a low-protein diet compared with those fed a high-protein diet (n > 20). Amino acid, aa. Red dashed line here and below shows the level of controls. (F) Overgrowth observed upon forced fat body expression of sun in larvae fed a low–amino acid diet is observed in mth1/+ but not in mth1/1 homozygous flies (n > 40). In graphs, means are shown, and error bars represent ±SEM; **P < 0.01. Renald Delanoue et al. Science 2016;353:1553-1556 Published by AAAS

Fig. 2 Mth and Sun control Dilp2 secretion from brain IPCs. Mth and Sun control Dilp2 secretion from brain IPCs. (A) Representative pictures of Dilp2 staining in IPCs (dashed outline) showing the kinetics of Dilp2 accumulation upon refeeding after prolonged starvation. Genotypes are as indicated. (B) sun silencing (lpp>sun-Ri) in the fat body prevents Dilp2 secretion upon refeeding, whereas sun overexpression (lpp>sun OE) reduces Dilp2 accumulation upon starvation. Graphs represent quantifications of ΔDilp2 fluorescence relative to lpp>w control after 90 min of feeding (means ± SEM; n > 20). Renald Delanoue et al. Science 2016;353:1553-1556 Published by AAAS

Fig. 3 Sun is a fat body–derived insulinotropic signal. Sun is a fat body–derived insulinotropic signal. (A) Hemolymph collected from fed, but not starved, larvae activates Dilp2 secretion when incubated on dissected brains from starved control (dilp2>w), but not mth-deficient (dilp2>mth-Ri), larvae. (B) Hemolymph from fed larvae deficient for adipose sun (lpp>sun-Ri) does not induce Dilp2 secretion. (C) Incubation of brains from starved larvae with various concentrations of SUN-A stimulates Dilp2 secretion. DMSO, dimethyl sulfoxide. (D) Hemolymph collected from fed larvae preincubated with preimmune serum (fed+PPI), but not from larvae treated with antibodies against SUN (αSUN), induces Dilp2 secretion. Titration of αSUN with blocking peptides allows reactivation of Dilp2 secretion. Diluted (dil.) blocking peptides do not block αSun action [fed+SUNpep(dil.) + αSUN]. Graphs represent quantifications of ΔDilp2 fluorescence relative to control brains (brains from starved larvae incubated with hemolymph, from fed larvae, or with DMSO) (means ± SEM; n > 20); **P < 0.01. Renald Delanoue et al. Science 2016;353:1553-1556 Published by AAAS

Fig. 4 The level of circulating Sun relies on TOR and PGC1 in fed animals. The level of circulating Sun relies on TOR and PGC1 in fed animals. (A) Circulating Sun peptide is detected by Western blotting of hemolymph from fed larvae (lanes 1 and 2) but not starved larvae (lanes 3 and 4). Antibodies against Crossveinless d lipoprotein (αCv-d) are used as a loading control. Quantification in arbitrary units (a.u.) of the normalized circulating Sun detected in the hemolymph according to nutritional conditions (means ± SEM; n = 4); **P < 0.01. (B) The sun transcript levels in the fat body decrease upon starvation and increase upon refeeding (measured by quantitative reverse transcription polymerase chain reaction) (means ± SEM; n = 4); **P < 0.01. (C) Circulating Sun levels decrease upon sun silencing in fat body cells (lpp>sun-Ri) but not in gut cells (myo1d>sun-Ri). Blocking TORC1 in fat cells (lpp>TSC1/2) strongly decreases circulating Sun. Quantification of normalized circulating Sun. (D) sun expression is severely reduced when PGC1 is silenced in fat body cells (means ± SEM; n = 3); **P < 0.01. (E) Silencing PGC1 in the fat body (lpp>PGC1-Ri) decreases pupal size (means ± SEM; n > 40); **P < 0.01. (F) Forced fat body expression of GFP-Sun (lpp>GFP-Sun) or Sun-GFP (lpp>Sun-GFP) rescues pupal size reduction observed from larvae fed the low–amino acid diet (means ± SEM; n > 40); **P < 0.01. Renald Delanoue et al. Science 2016;353:1553-1556 Published by AAAS