3h acetate

100 µM ³H-acetate (Perkin Elmer, Waltham, MA) was added to cells two hours prior to harvest. Cells were washed twice in 1× PBS and were lysed in 5% triton X-100. Following protein determination, cell lysates were extracted with chloroform/methanol (2:1) followed by addition of magnesium chloride to a final concentration of 1.64 mM. Following vortexing and centrifugation, the organic phase was extracted three times and dried at 70 °C, resuspended in 100% ethanol, and counted in scintillation fluid.

Cells were incubated overnight in Eagle's Minimum Essential Medium (EMEM) supplemented with 0.5% (w/v) fatty acid free bovine serum albumin. Then, cells were incubated for 4 h with fresh medium supplemented with 84 nM insulin and 20 μM acetate containing 20 μCi/ml [³H]acetate (Perkin Elmer, USA)58 (link). After the incubation, the cells were harvested, washed twice with ice cold phosphate buffered saline (PBS) (pH 7.4). Lipids were extracted and separated by thin layer chromatography. Lipid classes were visualized by exposure to iodine vapour, the corresponding bands were scraped, and the label incorporated into lipids was determined by scintillation counting and expressed relative to the cell protein.

BTEC cells were incubated 24 h with 1 µCi [³H]-acetate (3.6 mCi/mmol, PerkinElmer), then washed five times with PBS, and let grow for additional 48 h in fresh medium. After 72 h of radiolabeling, both cells and medium were collected. One aliquot of medium was used to generate the BTEC CM and incubated 24 h on LLC cells; the remaining part was used for the measurement of acetoacetate, as reported [21 (link)]. BTEC cells and LLC cells were collected by gentle scraping, re-suspended in PBS, and sonicated. A total of 100 µL cell lysates was used to measure the protein amount. A total of 300 µL of the lysates or of the medium were diluted 1:3 in the assay buffer (0.2 mM 3-(2-hydroxyphenyl) propionic acid, 10 mM p-Nitrobenzene diazonium fluoroborate solution, 0.4 M citrate buffer, pH 3.5, at 1:1:2 volume). The samples were incubated 5 min at 37 °C degree and resolved by an HPLC system equipped with a UV detector (SPD-20A) (Shimadzu, Kyoto, Japan), using the elution conditions described in [21 (link)]. The amount of [³H]-acetoacetate, an index of KBs synthesized by [³H]-acetate, was quantified according to calibration curves of serial dilutions of acetoacetate and expressed as µmol/mg cell proteins.

Differentiated WT and SSAO^−/− 3T3-L1 cells cultured in a 24-well plate were labeled with the trace levels (1.0 μCi) of [³H]acetate (Perkin Elmer) for 1 h, 2.5 h, and 4 h. Lipids were extracted with chloroform/methanol using the Folch method [43 (link)], and radioactivity was counted by liquid scintillation. Equal amounts of cells seeded in parallel in a 24-well plate were lysed with 1x RiPA buffer for protein content analysis using BCA assay kit (Pierce). The protein amounts of each well between WT and SSAO^−/− adipocytes were 151.2 ± 7.8 μg and 117.1 ± 2.1 μg (mean ± SD); the SD among wells is much smaller than the difference between WT and SSAO^−/− adipocytes. Radioactivity of cell lysates was counted in Ecoscint scintillation mixture (National Diagnostics) using a Beckman LS-6000 liquid scintillation counter. Radioactivity in each sample was normalized to protein content.

Rates of lipid synthesis and fatty acid beta-oxidation were assessed in PC-3 cells 24 h after transfection with the indicated siRNA. The lipid assay was based on the method of Lin and collaborators [54 (link)] with some modifications. Briefly, 24 h after transfection, PC-3 cells were incubated with [³H]acetate (2 μCi/ml; Perkin Elmer; used as a metabolic precursor of lipids) during 24 h Lipids were extracted using the chloroform–methanol method, and lipid radioactivity was measured by liquid scintillation (Lipoluma; Lumac). Triglycerides and phosphatidic acid content were measured in cells 48 h after transfection with the indicated siRNA. Lipids were extracted using the chloroform-methanol method. Triglycerides were measured by using a a colorimetric assay (Sigma, TR0100) and the phosphatidic acid content by using a fluorometric assay kit (Cayman chemical, 700240) following the instructions of the manufacturer.