2 nbdg

To assess insulin sensitivity, fat bodies (abdominal carcasses with the ovaries and digestive tract removed, n = 5 per sample) were dissected from live females anesthetized on ice and pooled into 1 mL Schneider’s medium (GIBCO 21720024). Samples were incubated for 15 min at 25°C, then treated ± 5 μM insulin (Sigma I9278) for a further 15 min at 25°C. The supernatant was removed and the fat bodies were homogenized into Laemmli buffer (BioRad #1610737) with 5% v/v β-mercaptoethanol. Samples were analyzed by western blotting, probing for phospho-AKT-Ser505 (1:1,000; Cell Signaling #4054) with total AKT (1:1,000; Cell Signaling #9272) and actin (1:1,000; Abcam #ab8224) as controls. Bands were analyzed by densitometry using FIJI software (ImageJ; Schindelin et al., 2012 (link)). To measure glucose uptake, dissected adult fat bodies (n = 5 per sample) were incubated in 100 μL Schneider’s medium with 200 μM 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; Invitrogen N13195) ± 5 μM insulin for 15 min at 25°C, protected from light. Samples were washed twice, then homogenized in 120 μL PBS using a pellet pestle and motor. 100 μL of supernatant was loaded in a 96-well plate, and the fluorescence (excitation/emission 485/520 nm) measured in a plate reader (FLUOstar Omega, BMG Labtech) against a 2-NBDG standard curve.

Glucose uptake was monitored by using a fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG, Cat# 11,046, Cayman Chemical Company, Ann Arbor, MI). Fully differentiated myotubes were treated with or without 5 μM imoxin in the absence or presence of 0.5 mM BSA-conjugated palmitate for 23 h. Cells were washed with sterile PBS and then stimulated with or without 50 nM insulin in glucose-free/serum-free DMEM for 30 min, followed by the addition of 2-NBDG at the final concentration of 100 μg/ml for an additional 30 min. The medium was then removed, and cells were washed twice with PBS and lysed with 0.5% Triton X-100 in 100°ul PBS to stop the further insulin stimulation. The fluorescent intensity of cellular 2-NBDG in each well was measured at excitation/emission wavelengths of 475 and 550 nm using a microplate reader (Fluostar Galaxy, BMG Labtech, Ortenberg, Germany).