Stable isotope tracing study design and sample collection

For each biological replicate, five whole flies per genotype at 1 week were fasted for 12 h prior to tracing experiment by placing them into empty plastic vials with cotton balls overnight. Then, a small aliquot (∼45 μl) of 30 mM [U-¹³C₆]glucose dissolved in diH₂O with blue dye was placed onto the side of the vials, which allowed flies to consume immediately. Immediate uniform consumption of the [U-¹³C₆]glucose mixture was verified using the blue dye indicator in gut tube. At 0.5, 1, 2, and 4 h for preliminary studies in Fig. S3(b), the flies were quickly exposed to 5 psi CO₂, placed into individual tubes at 5 flies per tube, and weighed so that samples can later be normalized to their weight. Tubes were placed into liquid nitrogen to snap-freeze the flies and stored in −80 °C until metabolite extraction was performed. For Fig. ​Fig.3,3, 1 week flies were offered 30 mM [U-¹³C₆]glucose for 1 h post fasting and collected as depicted above.