Priority Research Paper Volume 1, Issue 8 pp 699—713

Figure 2. Normal and reduced systemic insulin signaling as reflected by the cellular localization of the PH-tagged GFP reporter protein (tGPH) and dFoxO in fat body cells. (A-C) Increased UCP activities in the adult IPCs attenuate systemic insulin signaling events. In control flies, under normal growth conditions and a full strength PI-3' kinase activity, tGPH is predominantly located at the plasma membrane of each fat body cell (A). (B-C) UCP expression in adult IPCs results in a diffused, cytoplasmic distribution of the tGPH protein. Control: dilp2-Gal4, tGPH; mUCP1: dilp2-Gal4/UAS-mucp1, tGPH; hUCP2: dilp2-Gal4/UAS-hucp2, tGPH. (D-F) Increased accumulation of dFoxO in the nucleus of pericerebral fat body cells in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies indicates reduced insulin signaling. Cryosections of adult heads were stained with an α-dFoxO antibody followed by Alexa 568-conjugated secondary antibodies. A strong nuclear staining of the dFoxO protein was observed in the pericerebral fat body in both dilp2-Gal4/UAS-mucp1 (mUCP1, Panel E) and dilp2-Gal4/UAS-hucp2 (hUCP2, Panel F) flies but not in dilp2-Gal4/w1118 (Control, Panel D) flies. All sections were counter stained with DAPI (Panels G-I) to locate the nucleus of each cell. Merged images of anti-FoxO staining and DAPI are shown in Panels J-L. (M) Elevated levels of fasting circulating sugars are measured in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies. An average of 29% increase in circulating sugars measured in 14-day-old dilp2-Gal4/UAS-mucp1 (mUCP1) and dilp2-Gal4/UAS-hucp2 (hUCP2) females as compared to control dilp2-Gal4/w1118 (w1118) females. Each bar represents mean + SEM. N=5-7, *p= 0.046, **p= 0.05 (Student's t test).