Enzymatic kit

Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton® X-100, 0.1% SDS, 50 mM Tris, 0.5% Na-deoxycholate, pH 8) supplemented with a protease inhibitor cocktail (P8340, 1:1,000; Sigma–Aldrich, St. Louis, MO)) by 2 × 10 s sonication on ice. Protein concentration was determined by a Lowry assay (Bio-Rad Laboratories, Hercules, CA). Concentrations of TG, TC, and free cholesterol (FC) were measured with enzymatic kits (DiaSys, Holzheim, Germany), whereas CE concentrations were calculated by subtracting FC from TC.

Total serum and HDL lipids were measured using enzymatic kits from DiaSys Diagnostic Systems GmbH (Holzheim, BY, Germany). Total unesterified cholesterol in serum samples was determined by an enzymatic kit from Sigma Aldrich (St. Louis, MO, USA). Total HDL protein content was measured using BCA kit from Thermo Fischer Scientific Inc. (Waltham, MA, USA).

Serum glucose was assayed using enzymatic kits (DiaSys Diagnostic Systems GmbH, Holzheim, Germany). The serum free fatty acid concentration was assayed with a Roche FFA kit (Roche Applied Science). Serum glycerol was measured using the Free Glycerol Reagent (Sigma). Serum insulin, leptin and adiponectin concentrations were measured by radioimmunoassay (Millipore).

Blood samples were collected from mice. Plasma was separated by centrifugation, and cholesterol levels were measured using an enzymatic kit (catalog no.: 113009910026; Diasys Diagnostic Systems, Holzheim, Germany) with Cholesterol FS standard (catalog no.: 113009910030; Diasys Diagnostic Systems) for the calibration curve. Lipoprotein cholesterol distribution was measured by fast performance liquid chromatography using a system containing a PU-4180 pump with a linear degasser and UV-4075 UV/VIS detectors (Jasco, Tokyo, Japan). Pooled plasma samples (n = 15–16 mice per pool) were injected onto a Superose 6 Increase 10/300 GL column (GE Healthcare, Hoevelaken, The Netherlands) and eluted at a constant flow rate of 0.31 ml/min in PBS (pH 7.4). Cholesterol was measured in line by addition of cholesterol reagent at a constant flow rate of 0.1 ml/min using an additional PU-4080i infusion pump (Jasco, Tokyo, Japan). Data acquisition and analysis were performed using ChromNav software (version 1.0; Jasco, Tokyo, Japan).

Total lipids were extracted from kidney tissue according to Folch et al. [42 (link)]. The organic layer was dried under nitrogen gas and solubilized in isopropanol/Triton X-100 (10%) and then assayed for triglyceride concentration using an enzymatic kit (DiaSys). Data were expressed as the amount of triglycerides per gram of wet weight.

Placental villous tissue (50 mg) was homogenized in 1 ml acetone and total lipids were extracted by overnight incubation on a shaking platform. Samples were centrifuged at 16 000 g for 15 min, and 5 μl acetone extract was used to assay the TG concentration by an enzymatic kit (Diasys Diagnostic Systems) following the manufacturer's instructions.