(A) Fat bodies were isolated from WT and D/D adult flies (3 days), and lipid droplets were stained with Nile red. (B) Total triglycerides (TAG) were measured in adult flies (3 days) and normalized to total protein levels (32 flies per group). (C) Survival curves of starved flies (3 days) (160 flies per group). (D) Total TAG levels in adult flies (3 days) starved for various times (24 flies per group). (E) Tissue extracts were prepared from actin-GAL4/+ (act-Con) or actin-GAL4/UAS-dSH2B transgenic (act-dSH2B) adult flies (3 days), and immunoblotted with anti-dSH2B or anti-α-tubulin antibodies. (F) Whole body TAG levels were measured in actin-GAL4/+ and actin-GAL4/UAS-dSH2B adult flies (3 days) and expressed as % of actin-GAL4/+ TAG levels (32 flies per group). (G) Survival curves of starved flies (3 days)(120 flies per group). (H) Total TAG levels in starved adult flies (3 days) (32 flies per group). (I) Hemolymph carbohydrates in adult flies under fed conditions (60 flies per group). (J) The whole body carbohydrate levels in adult flies under fed conditions (32 flies per group). (K) Total head RNA was prepared from fed adult flies and used to measure dilp mRNA abundance (normalized to RPL32 expression) by quantitative RT-PCR (120 flies per group). *p< 0.05.

(A) S2 cells were transiently cotransfected with V5-tagged Chico plasmids and dSH2B plasmids. Cell extracts were prepared 48 h after transfection, immunoprecipitated with anti-V5 antibody, and immunoblotted with the indicated antibodies. Cell extracts were immunoblotted with anti-dSH2B or anti-V5 antibodies. (B) V5-tagged Chico was transiently co-expressed with or without dSH2B in S2 cells. Cells were treated with insulin for 15 minutes 48 h after transfection. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (C) S2 cells were incubated with dsRNA for 4 days, transfected with plasmids encoding V5-tagged Chico, and treated with insulin for 15 minutes. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (D) Chico mRNA abundance was measured by quantitative RT-PCR in wild type (WT) and Chico^C/C (C/C) adult flies (32 flies per group). (E) Total TAG levels were measured in adult flies (3 days) and normalized to total protein levels (32 flies per group). (F) Survival curves of adult flies (3 days) in response to starvation (120 flies per group). *p<0.05.

(A–B) Survival curves of wild type (WT) and dSH2B deficient flies (200 flies per group). (C–D) Survival curves of actin-GAL4/+ (act-Con) or actin-GAL4/UAS-dSH2B (act-dSH2B) flies (140 flies per group). (E–F) Survival curves of elav-GAL4/+ (elav-Con) or elav- GAL/UAS-dSH2B (elav-dSH2B) flies (180 flies per group). (G) Blood glucose levels in fed (44 weeks; WT: n=12, KO: n=10) and fasted (49 weeks; WT: n=12, KO: n=9) females. (H) WT (n=16) and KO (n=18) females were housed in a pathogen-free room, and their survivals were monitored daily. Survival rates were calculated as % of the initial number of mice. *p<0.05.

(A) A schematic representation of Drosophila and Homo sapiens SH2B proteins. PH: pleckstrin homolog domain; SH2: Src homolog 2 domain. The numbers indicate the similarity/identity, respectively. (B–C) dSH2B mRNA abundance (normalized to RPL32) in wild type w¹¹¹⁸ flies (> 400 embryos, >240 larvae, 40 pupae and 40 adult flies). L1: first instar, L2: second instar, L3: third instar lavae. (D) dSH2B mRNA abundance in wild type w¹¹¹⁸ adult flies under fed or starved conditions (n=4, 40 flies). (E) Total RNA was extracted from the whole body of dSH2B^D/D (D/D) and wild type (WT) adult flies (3 days) and reversely transcribed into cDNAs. dSH2B or β-actin cDNAs were amplified by PCR using dSH2B- or β-actin-specific primers. (F) Third instar larvae and adult flies. (G) The length of third instar larvae (WT and D/D: 64 flies) and the body weight of adult fly flies at 1 day of age (WT and D/D: 160 flies). (H) Adult fly Wings (3 days). Enlarged images are on the right. (I) Wing area and the size and total number of wing epithelial cells in adult flies at 3 days of age (n=32). *p<0.05.

(A–B) Adult flies (3 days) were randomly fed (F), fasted for 48 h (S), or re-fed for 24 h after 24 h starvation (R). The extracts were immunoblotted with the indicated antibodies. Akt phosphorylation was quantified by densitometry and normalized to total Akt protein levels (n=3). (C) Fat bodies were isolated from third instar larvae, and treated with or without human insulin (10 µg/ml) for 15 minutes. Cell extracts were immunoblotted with the indicated antibodies. (D) Fat body tissues were isolated from adult fly abdomens and treated with insulin (0.1, 1, and 10 µg/ml) as described in C. (E) S2 cells were transfected with dsRNAs against EGFP (si-EGFP) or dSH2B (si-dSH2B). dSH2B mRNA abundance was measured by quantitative RT-PCR 4 days after transfection (n=4). (F) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) for 15 min. Cells extracts were immunoblotted with the indicated antibodies. (G) V5-tagged dFOXO was transiently coexpressed with or without dSH2B in S2 cells, and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) as described in F. (H–I) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (10 µg/ml) for 15 min. Cells were immunostained with anti-V5 antibody and visualized using a confocal microscopy. Cytoplasmic dFOXO-positive Cells were counted and normalized to the total number of cells (four independent experiments). *p<0.05.

(A, D, G) Whole body TAG levels were measured in adult flies (3 days) and normalized to total protein levels (32 flies per group). (B, E, H) Hemolymph sugar levels in adult flies (3 days)(60 flies per group). (C, F, I) Survival curves of adult flies (3 days) in response to starvation (100 flies per group). *p<0.05.

(A–C) Adult flies (3 days) were treated with 30 mM paraquat, and survival rates were measured (120 flies per group). (D) Wild type (n=6) and KO (n=6) females (11–14 weeks) were intraperitoneally injected with paraquat (70 mg/kg body weight), and survivals were monitored hourly. Survival rates were calculated as % of the initial number of mice.