Lipoprotein lipase

As previously done^{67 (link)}, flies were homogenized in 500 µL of PBST (0.05% (v/v) Tween 20 in PBS) using a plastic pestle motor mixer. 200 µL of lysate was incubated at 70 °C for 5 min, chilled on ice, and incubated with 1 µL of 25 KU/mL lipoprotein lipase from Chromobacterium viscosum (Calbiochem) at 37 °C overnight. Debris was removed by centrifugation at 14,000 rpm for 3 min. Triglyceride content was determined by mixing 15 µL of the supernatant with 150 µL Free Glycerol Reagent (Sigma), incubating at 37 °C for 6 min, and measuring the absorbance at 540 nm. Triglyceride content was normalized to total protein levels measured by Bradford assay at 595 nm. To measure triglyceride levels in the fat body, flies were dissected in cold PBST, and collected fat bodies were processed with same procedures, as described above. For determining glycogen levels, 30 µL of the supernatant (after lipoprotein lipase treatment) was treated with 14 Units of amyloglucosidase (Sigma) at 50 °C for 1 h. 15 µL of the treated mix was combined with 150 µL of glucose reagent (Sigma), incubated at 37 °C for 30 min, and absorbance was measured at 340 nm. Glycogen content was also normalized to total protein. Triplicates of 10 flies/age group were used.

The buprenorphine (BUP) prodrugs were synthesised from BUP (Tasmanian Alkaloids, Tasmania, Australia) as described in the Supplementary Material. Oleic acid, Tween 80, lipoprotein lipase, and porcine pancreatin were purchased from Sigma-Aldrich, MO, United States. Acetonitrile for liquid chromatography was purchased from Merck Pty Limited, VIC, Australia. Ultrapure water was obtained from a Milli-Q™ system (Millipore, MA, United States). All other chemicals were analytical grade or above. Lipid-based formulations of the prodrugs or BUP for in vivo studies were assembled as described previously (Han et al., 2014 (link)) using ultrasonication. Formulations for each animal in the lymphatic transport studies contained 50 µg of prodrug or parent BUP, 40 mg Oleic acid, 25 mg Tween 80 and 5.6 ml phosphate buffered saline (PBS, pH 7.4). Formulations for each animal in the oral bioavailability studies contained 50 µg of prodrug or 20 µg of parent BUP, 40 mg Oleic acid, 25 mg Tween 80 and 2 ml PBS. The IV formulation was a solution of 20 μg/ml BUP in PBS (10 µg in 0.5 ml for each animal).