Fig 2.

Effect of tissue-specific overexpression of mth on lifespan and stress resistance (A) Localization of mth transgene in IPCs, including the axons which carry the secreted peptides to the periphery. (B) Relative mRNA abundance in the transgenic flies increased 12–14 fold compared to the mean of three control lines at 5 days of age based on real-time quantitative PCR. (C & D) Survival of transgenic flies (n = 230–330 per survival experiment) compared with controls. Flies were maintained at 25°C, 12/12 h dark/light cycle and were transferred to fresh food vials every 3–4 days. Mean longevity of males was 74, 79, and 77 days for the three control strains compared with 86 and 93 days for mth22 and mth25 lines, respectively. For females, the analogous longevities were 85, 79, and 81 days for the same control strains compared with 93 and 99 days for mth22 and mth25, respectively. (E) Stress resistance to paraquat in mth overexpressing flies. Newly eclosed flies were sex-segregated and maintained in fresh food vials for 5 days before testing. Then flies were starved for 6 h and transferred to vials containing Kimwipes wetted with 20mM paraquat in 5% sucrose solution, and monitored for survival at 25°C. Data shown are survival at 36 h post-paraquat exposure. Data for three control lines has been combined. Relative abundance of mRNAs of three Dilps produced by the IPCs in heads (F) and bodies (G) of transgenic lines compared to combined values of three control lines as measured by RT-PCR. As with the IPC-mthRNAi lines, Dilp2 and Dilp5 were significantly affected, whereas Dilp3 was not. (H) Glucose concentration in hemolymph of both transgenic lines is significantly higher than in control lines. (I) Direct observation of GFP-tagged Dilp2 release in IPCs of control (w¹¹¹⁸ x UAS-mth618) and mth overexpressing lines (UAS-mth618 x Dilp2-GFP-Gal4) showing ~40% reduction in DILP2 release over 15 minutes in the transgenic line. DILP release was stimulated as in Figure 1. (J) Effect of IPC-specific mth overexpression on β-arrestin levels in the brains of 5 day old flies. Relative abundance of all RT-PCR is shown have been normalized to control which is shown as dotted line as one. In all other experiments: Control 1 = w¹¹¹⁸ x Dilp2-GAL4, Control 2 = w¹¹¹⁸ x UAS-mth22-GFP, Control 3 = w¹¹¹⁸ x UAS-mth25-GFP; mth22 = UAS-mth22-GFP x Dilp2-GAL4; mth25 = UAS-mth25-GFP x Dilp2-GAL4.