13C-isotopic labeling experiments and mass isotopomer analysis

To keep the molar ratio of the labeled glucose to sucrose consistent, we prepared the normal and high sugar diet with 13 mM and 50 mM of [U-¹³C₆]glucose, respectively. For ¹³C-isotopic labeling experiments, we collected 2 – 4 days old w¹¹¹⁸ male flies and placed approximately 30 flies in vials containing either NSD or HSD with isotope tracers at room temperature. The food was replaced every two days. On day five, twenty thoraces were dissected per biological replicate and intracellular metabolites were extracted using 80% (v/v) aqueous methanol. Q1/Q3 SRM transitions for incorporation of 13C-labeled metabolites were established for polar metabolite isotopomers, and data were acquired by LC-MS/MS. Peak areas were generated using MultiQuant version 2.1 software. The natural isotope abundance was corrected using AccuCor (Github: https://github.com/XiaoyangSu/AccuCor)⁷⁷.