Infinity glucose hexokinase reagent

Flies were homogenized in 200 μl of cold trehalose buffer (5 mM Tris-HCl, pH 6.6, 137 mM NaCl and 2.7 mM KCl). The homogenate was heat-treated at 70 °C for 5 min to inactivate endogenous enzymes. Samples were centrifuged for 3 min at 16,100 × g and the supernatant collected. Protein concentration was measured using a Bradford assay (Bio-Rad 5000006). Samples were diluted with trehalose buffer and 90 μl of heat-treated homogenate were incubated with either 20 μl of trehalase (Sigma-Aldrich T8778) or 20 μl of trehalose buffer. To create a trehalose (Sigma-Aldrich T9531) standard curve, 50 μl of standard were incubated with either 30 μl of trehalase or 30 μl of trehalose buffer. A free glucose (Fisher Scientific 50-99-7) standard curve was also generated. All samples were incubated at 37 °C overnight. Then, 30 μl of each sample was added to a 96-well plate. To all samples, 100 μl of Infinity Glucose Hexokinase reagent (Thermo TR15421) was added and incubated at room temperature for 15 min. The absorbance of samples was then measured at 340 nm and normalized by subtracting the absorbance of free glucose present in untreated samples from the total amount of glucose present in samples treated with trehalase. Trehalose and free glucose content were calculated based on standard curves of trehalose and glucose respectively.

Glucose concentration in Drosophila hemolymph and brains was measured using the Infinity Glucose Hexokinase reagent (Thermo scientific). To measure glucose in the hemolymph, samples of 10 larvae were washed in NaCl 0.7%, 0.1% Triton X-100 and then in d-H₂O. The hemolymph of these larvae was collected and its glucose content measured following the protocol of Rulifson et al. [38] (link). The values reported in Figure 2A are the means ±SE of 8 samples of 10 larvae. To measure glucose in brains, samples of 20 brains were placed in 40 µl of 10⁻³ M EDTA, 10⁻²M KH₂PO₄, and the complete protease inhibitor cocktail (Roche), mechanically homogenized and then centrifuged at 14,000 rpm for 10 min. The supernatant was collected with a micropipette and used for glucose measurement according to Rulifson et al. [38] (link). The measures reported in Figure 2B are the means ±SE of 4 samples of 20 brains.
Insulin stimulation of Akt phosphorylation was performed using recombinant human insulin (Sigma, I0516). Before stimulation, larvae were starved for 5 hours in an empty vial humidified with a drop of saline devoid of FBS. We then followed the protocol described by Musselman et al. [41] (link). However, at the end of the insulin stimulation procedure, instead of larvae, we homogenized samples of 20 isolated brains, which were then used for Western blotting analysis.

Glucose concentration in hemolymph from third instar larvae was measured using the Infinity Glucose Hexokinase reagent (Thermo scientific). Hemolymph collection and glucose measurement were done as described in Marzio et al.^{14 (link)}.