Nefa hr 2 assay

Glucose, lipoproteins, and alanine aminotransferase (ALT) in plasma and glycated hemoglobin (HbA1C) in whole blood samples (collected in EDTA tubes) were quantified by Cobas c111 analyzer (Roche Diagnostics, Mannheim, Germany). A sensitive rat RIA kit (EMD Millipore, Billerica, MA, USA) was used to measure insulin. NEFA-HR2 assay (Wako Chemicals GmbH, Neuss, Germany) was utilized to determine non-esterified fatty acids (NEFAs) concentration. Plasma total antioxidant capacity was quantified by a commercial assay kit (MAK 187, Sigma Aldrich, Steinheim, Germany) according to the manufacturer’s instructions. Liver triglycerides content was estimated by cutting around 50 mg of frozen liver tissue per sample on dry ice and obtaining its saponified extract using a published protocol [25 (link)]. Triglyceride concentration was then quantified by a Cobas c111 analyzer.

Lipolysis was assessed in differentiated adipocytes under basal conditions and after the addition of CL or ISO (final concentrations of 100 nM) for 3 h. Glycerol release was measured using a lipolysis assay kit (ab185433, Abcam) according to the manufacturer's protocol. Free fatty acids (FAs) were measured in supernatants of differentiated adipocytes stimulated with CL or ISO (final concentrations 1 μM or 100 nM) for 3 h according to manufacturer's protocol (NEFA‐HR(2) Assay, Fujifilm Wako Chemicals Europe, Neuss, Germany). Supernatants of differentiated brown adipocytes, pre‐treated with or without 100 nM or 5 μM CL for 60 min, were collected and cellular l‐lactate release was measured using standard colourimetric assay as described in the manufacturer's protocol (ab65331, Abcam). Intracellular superoxide and ROS levels in differentiated brown adipocytes, pre‐treated with or without 1 μM CL for 60 min, were measured using a standard assay kit (ab139476, Abcam).

Blood samples were taken one day after hospital admission from the fasting patient for subsequent biomarker determination. Serum and Ethylenediaminetetraacetic acid (EDTA) plasma was prepared by centrifugation at 1452 g and 5 °C for 10 min and were frozen at −80 °C till analyses. NEFA were assessed in serum samples with Pentra C400 benchtop analyser (Horiba Medical, Grabels, France) using NEFA-HR (2) Assay (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) according to the manufacturer’s instructions.
In addition, on the day of hospital admission, the following laboratory parameters were routinely determined in all patients: blood count, electrolytes, renal retention parameters (estimated glomerular filtration rate, eGFR according to Chronic Kidney Disease, CKD-EPI formula, serum creatinine), lipid status (total cholesterol, LDL and HDL cholesterol, triglycerides), C-reactive protein (CRP), NT-Pro-BNP, blood glucose and HbA1c. For routine blood measurements the autoanalyzer COBAS Integra 800 (Roche Diagnostics, Basel, Switzerland) was used.

Slaughter blood samples were collected immediately after sacrifice and allowed to coagulate at room temperature. After centrifugation of liquid phase (10 min, 1,500 g, 4°C), serum was collected, frozen and stored until analysis. Serum levels of non-esterified fatty acids (NEFA), triglycerides and glucose were determined with the automatic enzymatic analyzer ABX Pentra 400 (Horiba Medical) as described before^{55 (link),104 (link)} using commercial kits (NEFA-HR(2) Assay, WAKO Chemicals; Pentra Triglycerides CP, Axonlab AG; Pentra Glucose HK CP, Axonlab AG). The detection limit and inter-assay CV were as follows: NEFA 0.11 mmol/l, 0.9%; triglycerides 0.08 mmol/l, 1.4%, glucose 0.11 mmol/l, 0.9%.

Adipocytes were cultured in 96-well plates and the release of glycerol and free fatty acids into the medium was measured on day 8. Cells were washed twice with respiration base medium and respiration assay medium was added to a final volume of 180 μl per well (inhibitors were added to the assay medium at this point and were present in the medium during the measurement, see 2.8). The cell culture plate was incubated for 1 h at 37 °C in a laboratory non-CO₂ incubator. After that, isoproterenol, oligomycin, or a combination of both was added to the medium. The cell culture plate was placed back into a laboratory non-CO₂ incubator and incubated at 37 °C. After an additional hour, conditioned media were harvested and stored for further analysis. Free glycerol was determined with the Free Glycerol Determination Kit (Sigma–Aldrich) and free FAs were quantified with the NEFA-HR(2) Assay (FUJIFILM Wako Chemicals). Free FA re-esterification rate was calculated as previously described [10 (link),30 (link)]. Free FAs re-esterified = 3 ∗ glycerol – free FAs.

Whole blood glycated hemoglobin (HbA_1C) (collected in EDTA tubes), and plasma glucose, lipoproteins, and alanine aminotransferase (ALT) were quantified by Cobas c111 analyzer (Roche Diagnostics, Mannheim, Germany). A sensitive rat RIA kit (EMD Millipore, Billerica, MA, USA) was used to measure insulin, and a NEFA-HR2 assay (Wako Chemicals GmbH, Neuss, Germany) was performed to determine non-esterified fatty acids (NEFAs) concentrations. Plasma adiponectin was quantified by AssayMax ELISA kit (Assaypro, St. Charles, MO, USA) and IL1b by Cloud-Clone Corp. (Houston, TX, USA). For liver triglyceride content measurement, 50 mg of frozen liver tissue per sample was weighed on dry ice, and its saponified extract was extracted using a published protocol [18 (link)]. Triglyceride levels were then quantified by Cobas c111 analyzer.
Caffeic acid, trigonelline hydrochloride, cafestol acetate, and ferulic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) and isotope-labeled hippuric acid (Ring-13C6, 99%) from ReseaLife Chem Science (Burgdorf, Switzerland). HPLC-grade acetonitrile, methanol and acetic acid were purchased from Fluka (Sigma-Aldrich, St. Louis, MO, USA).

Plasma insulin levels were measured using mouse/rat insulin ELISA (Crystal Chem., Downers Grove, USA). Plasma FFAs were measured by NEFA-HR(2) Assay (WAKO Chemicals, Neuss, Germany). Liver and muscle DAGs were measured by liquid chromatography-mass spectrometry/mass spectrometry in the lab of Gerald I. Shulman (Yale University, USA) as described previously [77 (link)]. Hepatic TAG content was determined using a triglyceride assay (DiaSys, Holzheim, Germany) following the manufacturer's instructions. Inflammation markers in plasma samples were analyzed by electrochemiluminescence assays (Meso Scale Discovery, Rockville, USA).