F6428

For measurement of the whole-body triglyceride (TAG) content, 20 flies per group were homogenized in 0.5 mL PBS. After sufficient mixing of 0.4-mL homogenates with 1.6 mL of CHCl3:CH3OH (2:1, vol/vol), the suspension was centrifuged at 2,500 rpm. for 10 min at room temperature. The lower organic phase was transferred and air-driedovernight in a chemical hood. The residual liquid was re-suspended in 0.5 mL of 1% Triton X-100 in absolute ethanol, and the concentration of TAG was determined using the serum triglyceride determination kit (triglyceride reagent T2449 and free glycerol reagent F6428, Sigma-Aldrich, MO, USA).

We used a modified version of a well-established lipolysis assay [36] (link) to evaluate NEFA, glycerol, and leptin release from adipose organ explants. Briefly, eWAT, iWAT, and BAT from Adipoq-GsD, Adipoq-GiD and respective littermate controls were surgically removed and cut in ∼20 mg pieces. The pieces were put in a 96-well plate containing 200 μl of prewarmed (37 °C) DMEM no phenol red (Gibco A1443001). The pieces were then incubated at 37 °C (5% CO2, and 95% humidified atmosphere) for one hour (baseline) in DMEM containing 2% fatty acid (FA)-free bovine serum albumin (BSA, Sigma, A8806) and 0.1% glucose (Sigma 49163). To avoid re-esterification of FA and glycerol, each well also contained 5 μM of the acyl-CoA synthetases inhibitor triacsin C (Tocris 2472). At the end of the hour, the pieces were transferred to a new 96-well plate containing fresh medium with 1 μM CNO (C0832, Sigma) and incubated for another hour (stimulated). The incubation media from baseline and stimulated conditions were then used to evaluate NEFA release by colorimetry (FUJIFILM Wako Diagnostics-NEFA Reagent, 999-34691, 995-34791, 991-34891, 993-35191), glycerol release by colorimetry (F6428, Sigma), and leptin release by ELISA (Mouse/Rat Leptin ELISA, ALPCO, 22-LEPMS-E01). NEFA, glycerol, and leptin release were then calculated based as a percentage compared to baseline.