Ab108785

In order to measure adiponectin in cell culture media, an adiponectin mouse ELISA kit was used according to the manufacturer's instructions (ab108785, Abcam, Cambridge, UK). Concentration was normalized to total protein content.

To determine insulin clearance at a steady state, mice were fasted from 1700 h until 1100 h the next day, and their retro-orbital venous blood was drawn into heparinized micro-hematocrit capillary tubes (Fisherbrand, Waltham, MA, USA), as previously optimized [6 (link)]. Plasma was processed and analyzed for insulin (80-INSMSU-E01, Alpco, Salem, NH, USA), C-peptide (80-CPTMS-E01, Alpco), adiponectin (ab108785, Abcam, Cambridge, MA, USA), non-esterified fatty acids (NEFA-C colorimetric assay; Wako, Richmond, VA, USA), tumor necrosis factor-alpha and interleukin-6 (ELISA Kits, Abcam), and nitric oxide (NO) (Nitrate/Nitrite fluorometric assay Kit; Cayman Chemical, Ann Arbor, MI, USA). To determine plasma leptin levels, mice were fasted up to 4 h in the morning before blood was drawn (Linco Research, Billerica, MA, USA).

Whole animal or human blood samples were separated by being allowed to sit for 30 min or using EDTA-coated tubes, followed by centrifugation at 4000 ×g for 15 min and storage at -80°C until use. Blood glucose, triglycerides, total cholesterol, glucose, and AST/ALT were measured using Fuji Dri-Chem Slides (Fujifilm). Leptin, ghrelin, GLP-1, adiponectin, and glucagon concentrations were determined in duplicate according to the manufacturer’s ELISA instructions. ELISA kits for mouse adiponectin and leptin were purchased from Abcam (ab108785 and ab100718; Cambridge, UK), and ELISA kits for mouse ghrelin, GLP-1, and glucagon were from Cusabio (CSB-E09817m, CSB-E08118m, and CSB-E15775m, respectively; Houston, TX). Enzyme immunoassay (EIA) kits for human/mouse apelin-13 were purchased from Phoenix Pharmaceutical (EK-057-19, Burlingame, CA). ELISA kits for glucose (Ab6533, Abcam), triglycerides (ETGA-200, Bioassay Systems, Hayward, CA, USA), and cholesterol (ab65390, Abcam) were used to measure levels in the liver tissues and sera saved after the experimental diets and from control and NAFLD patients.

Blood was collected from mice at the age of 24 weeks fasted for 4 h. Fasting serum adiponectin (cat. no. ab108785, Abcam), leptin (cat. no. DY498, R&D), free fatty acid (cat. no. LabAssay 294-63601, Wako), ethanol (cat. no. ab65343, Abcam), norepinephrine (cat. no. ab287789, Abcam), insulin (cat. no. 10-1247-01, Mercodia), and 4-HNE (cat. no. EEL-M2677, Elabscience) concentrations were measured using enzymatic or ELISA assay kits according to manufacturer’s instruction. Plasma triglycerides, and total cholesterol levels were measured using the FUJI DRI-CHEM clinical chemistry analyzer.