Nefa hr 2

We used glucose (catalog 8769), glycerol (catalog G2025-1L), poloxamer 407 (catalog 16758-250G), and dextran sulfate (catalog D8906-5G) from MilliporeSigma; Ensure Original Nutritional Shake from retail pharmacy; PHA-665752 (catalog 14703) from Cayman Chemical; mouse recombinant active HGF protein (catalog 2207-HG) from R&D Systems, Bio-Techne; mouse Ultra-Sensitive Insulin ELISA from Crystal Chem (catalog 90080); Triglyceride LiquiColor test (catalog 2200225) from StanBio Laboratories; total cholesterol (catalog 999-02601) and NEFA-HR(2) (Wako); and thrombin (catalog T4648-1KU) from MilliporeSigma.

Capillary blood glucose was measured using the glucose oxidase method (Dr. Müller Super GL, Freital, Germany). Triglycerides, cholesterol, LDL- and HDL-cholesterol, CRP, and liver enzymes were measured by standard laboratory methods using Cobas ISE direct and c111 Analyzer (Roche Diagnostics, Mannheim, Germany) [23 (link)]. Serum insulin was measured by fluoroimmunometric assay (AutoDelfia; Perkin Elmer, Rodgau, Germany) (inter-assay CV 2.3–3.5%, intra-assay CV 1.7–2.4%). Non-esterified fatty acids (FFA) were quantified in serum using a commercially available colorimetric assay (NEFA HR2, Wako, Neuss, Germany) performed on ABX Pentra 400 (HORIBA ABX, Montpellier, France) (inter-assay CV < 5%, intra-assay CV < 1%). Fetuin-B was assessed in serum samples by ELISA using a commercial ELISA kit (Human Fetuin B DuoSet ELISA; R&D Systems, Minneapolis, USA), following the manufacturer’s protocol (intra-assay CV: 5.3%, inter-assay CV: 8.2%). Plasma fetuin-A was also determined by an ELISA method (BioVendor Laboratory Medicine, Brno, Czech Republic (intra-assay CV: 3.6%, inter-assay CV: 4.5%)) in accordance with the manufacturer’s instructions.

LPL activity was in principle measured through detecting free fatty acid (FFA) concentration [22 (link)]. The reaction substrate, termed buffer A, was composed of 1 ml TG-rich serum (TG concentration, > 3000 mg/dL) from Gpihbp1-deficient mice (Gpihbp1^−/−) [23 (link)], 0.18 g 10% fatty acid-poor bovine serum albumin (BSA) (Miles, West Haven, CT), 0.031 mg heparin, 0.012 g NaCl and 0.3 mmol Tris-HCl Buffer (pH 8.5), in a final volume of 5 mL. 5 μL buffer A were mixed with 5 μL serum from wild-type rats and 5 μL test sample, and incubated at 37 °C for 60 min. [Note that serum from either Gpihbp1-deficient mice or wild-type rats was pre-incubated for 10 min at 62.5 °C in order to inactivate any residual endogenous lipase activity.] FFA concentration was determined in triplicate on a spectrophotometer (Thermo Multiskan GO) using the Wako kit, NEFA-HR(2).
In the case of human serum test sample, the FFA concentration represented the total post-heparin lipase activities that comprised LPL and hepatic lipase (HL) activities. To correct for the contribution from HL, 1 M NaCl was added and incubated for 60 min, so that the LPL activity can be completely inhibited [24 (link)]. LPL activity was then calculated by the difference between total post-heparin lipase activity and HL activity. All assays were performed in triplicate.

Fasting plasma glucose was measured using a hexokinase method on an autoanalyser (Roche Diagnostics Hitachi 917, Hitachi Ltd., Tokyo, Japan). Serum insulin concentration was determined by time-resolved fluoroimmunoassay using Insulin Kit (AUTOdelfia, Wallac, Turku, Finland). HbA_1C was measured by high-pressure liquid chromatography using a fully automated Glycosylated Hemoglobin Analyzer System (BioRad, Richmond, CA). Plasma total and HDL cholesterol and triglyceride concentrations were measured with respective enzymatic kits from Roche Diagnostics using an autoanalyzer (Roche Diagnostics Hitachi 917, Hitachi Ltd., Tokyo, Japan). Serum FFA concentration was measured by an enzymatic colorimetric assay (NEFA-HR(2), Wako Chemicals GmbH, Neuss, Germany) using a Konelab 60i analyzer (Thermo Electron Corporation, Vantaa, Finland). Plasma ALT, AST, GGT and creatinine concentrations were determined as recommended by the European Committee for Clinical Laboratory Standards.