Nefa c test kit

Plasma glucose was determined with the glucose oxidase method using a Biosen C-line plus glucose analyzer (EKF Diagnostics, Barleben/Magdeburg, Germany). Plasma insulin was determined by immunoassay on an Atellica system (Siemens, Erlangen, Germany) with intra-assay variation of 3% and inter-assay variation of 7%. Plasma FFAs were determined by an enzymatic colorimetric method (NEFA C test kit; Wako Chemicals, Neuss, Germany) with intra-assay variation of 1% and inter-assay variation of 4–15%. Plasma glucagon was determined by radioimmunoassay (Linco Research, St Charles, MO, USA) with intra-assay variation of 4–8% and inter-assay variation of 6–11%. Plasma leptin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay and inter-assay variation of 6%. Plasma ghrelin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay variation of 4% and inter-assay variation of 6%.

Frozen liver and WAT were thawed, homogenized in PBS, and mixed with a solution of chloroform/methanol (2:1). The mixture was swirled on a rotating at 4 °C for 12 h, and centrifuged at 3,000 x g for 15 min. The lower layer was obtained and evaporated at 40 °C to dryness under a stream of nitrogen gas. The residue was dissolved in ethanol, and tissue contents of TC, TG, NEFA and glycerol were measured using Determiner L TC II kit (Kyowa Medex, Tokyo, Japan), L-Type TG M kit (Wako, Osaka, Japan), NEFA C-test kit (Wako, Osaka, Japan) and glycerol colorimetric assay kits (Cayman chemical, Michigan, USA), respectively, following the manufacturer’s instructions.

A portion of liver tissue from each mouse was used for analyzing TG and total TC contents. Hepatic lipids were extracted from approximately 100 mg of liver tissue for each mouse in accordance with the method of Folch et al.38 (link). Quantification of liver and plasma TG and TC were performed using the Triglyceride E-Test and Cholesterol E-Test kits, respectively, while plasma NEFA were quantified using the NEFA C-Test kit (Wako Pure Chemical Industries, Ltd). Plasma leptin levels were quantified by enzyme-linked immunosorbent assay (ELISA) using the Leptin/mouse ELISA kit (Morinaga Institute of Biological Science, Tokyo, Japan). FGF-21 levels in plasma were measured with the FGF-21 ELISA kit (R&D System, Minneapolis, USA).

Serum alanine aminotransferase (ALT) was measured using Spotchem SP-4410 (Arklay Co., Kyoto, Japan). The total serum cholesterol (Chol) and triglyceride (TG) levels were analyzed using an online dual-enzymatic method for the simultaneous quantification of cholesterol and TG using high-performance liquid chromatography according to a previously described procedure [4 (link), 9 , 10 (link)]. The fasting plasma glucose level and the serum levels of fasting insulin, leptin, adiponectin, and non-esterified fatty acid (NEFA) were determined using a Glutest Pro kit (Sanwa Kagaku Kenkyusyo Co., Nagoya, Japan), an Ultra Sensitive Insulin ELISA kit (Biochemical Research Laboratory, Morinaga Milk Industry Co., Tokyo, Japan), an ELISA Mouse Leptin kit (Biochemical Research Laboratory, Morinaga Milk Industry Co., Tokyo, Japan), a Mouse/Rat Adiponectin ELISA kit (Otsuka Pharmaceutical Co., Tokyo, Japan), and a NEFA C-Test kit (Wako Pure Chemical Industries Co., Osaka, Japan), respectively. The blood IR was estimated using the homeostasis model assessment of IR (HOMA-IR) and the following equation: IR = fasting plasma glucose level (mg/dL) × fasting serum insulin level (ng/mL)/22.5.

Plasma insulin was determined with an Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Inc.) following the manufacturer’s protocol. Plasma FFA was measured with a NEFA C Test Kit (Wako Chemicals, Richmond, VA). Plasma triglycerides were analyzed using plasma Triglyceride Determination Kit (Sigma-Aldrich, St, Louis MO) and plasma cholesterol was determined by a Cholesterol/Cholesteryl Ester Quantitation Kit (BioVision, Milpitas, California). Plasma IL-6 and myostatin levels were measured by using ELISA Kit (EMD Millipore). Plasma irisin was determined using a mouse irisin ELISA Kit (Phoenix pharmaceutical, Burlingame, CA).