Trehalase solution

For TG assay, flies were homogenized in 300 µl of 0.05% PBS-Triton, and the supernatant was heated for 10 min at 70°C to inactivate endogenous enzymes. 300 µl of Infinity TG Reagent (Thermo Electron Corp) was added to 20 µl homogenate and incubated at 37°C for 15 min. TG levels were measured as the OD value at 520 nm in Spectra Max 340 Microplate Reader and total amounts were determined using TG standard. For trehalose assay, flies were homogenized in a buffer containing 5 mM Tris pH 6.6, 137 mM NaCl and 2.7 mM KCl, followed by a heat treatment for 10 min at 70 °C to inactivate endogenous trehalase. Half of the supernatant was diluted with an equal volume of the buffer to provide the basal value, the other half was diluted with trehalase solution (Sigma) to break trehalose into free glucose at 37 °C for 20 hours, and glucose levels were measured using Glucose (HK) Assay Kit (sigma) via reading of OD value at 340 nm. To measure the circulating trehalose, hemolymph was extracted by decapitation and centrifugation from 30–50 adult flies, and an aliquot of collected hemolymph was used to measure trehalose using the assay described above.