Bio safe 2

After the 12-week experimental period, mice were fasted for 16 h, anesthetized with sodium pentobarbital (50 mg/kg; P3761, Sigma), and sacrificed. Epididymal WATs were harvested, preincubated in KBH buffer (8 mM glucose, 5 mM HEPES, 0.1% bovine serum albumin (fraction V), and 2 mM sodium acetate) at 30 °C for 20 min, and then incubated in KBH buffer containing 0.3 μCi [¹⁴C] mannitol and 2 μCi/mL 2-[³H]deoxy-glucose without 100 nM human insulin in a shaking water bath at 30 °C for 30 min. The tissues were blotted, rapidly freeze-clamped, and weighed before being dissolved in 0.5 mL of 1 M KOH for 20 min at 70 °C and neutralized with 0.5 mL of 1 M HCl. A 0.3 mL aliquot of each sample was mixed with 6 mL of Biosafe II (Research Products International, Mount Prospect, IL, USA) and radioactivity quantified using a liquid scintillation counter. The [¹⁴C] mannitol concentration was measured to quantify extracellular 2-[³H] deoxy-glucose and glucose uptake was calculated by subtracting extracellular 2-[³H] deoxy-glucose from total 2-[³H] deoxy-glucose.

Extracellular PPi levels were measured using the differential adsorption of uridine diphospho-(6-³H) glucose (GE Healthcare, Buc, France), and its reaction product 6-phospho-(6-³H) gluconate on activated charcoal, as previously described [44 (link)]. The standard concentrations, ranging from 10 to 400 pmol of ePPi, were included in each assay. After adsorption of the reaction mixture on charcoal, and centrifugation at 14,000 rcf for 10 min, 100 μL of supernatant were removed carefully and counted for radioactivity in 5 mL of Bio-Safe II (Research Products International Corp., Mt. Prospect, IL, USA). Results were expressed as picomoles of ePPi per microgram of total cell proteins.

80 µl solutions containing His-tagged NPC1L1 N-terminal domain or Domain2 (150 nM) were incubated with cholesterol (5 µM, 1:1000 ³H:¹H) in Buffer A (50 mM HEPES (pH 7.4), 150 mM NaCl), containing either 0.004% NP-40 and BSA (192 nM) or mixed bile salt micelles for 30 min at 37°C. Micelle lipids were first dissolved in ethanol, combined in a glass vial, and solvents evaporated under N₂. The dried lipids were incorporated into buffer A containing 24 mM sodium taurocholate by vortexing for 5 min. The solution was then diluted using buffer A to final concentrations: 5 mM sodium taurocholate, 0.5 mM oleic acid, 0.035 mM phosphatidylcholine, 0.08 mM lysophosphatidylcholine, 0.3 mM monoolein, 0.005 mM cholesterol, and 5 nM radiolabeled cholesterol (Johnson and Pfeffer, 2016 (link)). After incubation, solutions were loaded onto 1 ml syringes fitted with a frit and 20 µl Ni-NTA resin. After 15 min, the resin was washed 6X with 1 ml wash buffer (buffer A + 10 mM imidazole + 0.004% NP-40) and eluted with 1.2 ml elution buffer (buffer A + 250 mM imidazole + 0.004% NP-40). Eluted samples were mixed with BioSafe-II (Research Products International, Mt. Prospect, IL), and radioactivity was determined using a scintillation counter.